Metalloproteinase-disintegrin polypeptides and methods of making and use thereof

ABSTRACT

Provided is a new disintegrin polypeptide, methods of making such polypeptides, and methods of using them to treat disintegrin-associated disorders and conditions and to identify agents that modulate Metalloproteinase-Disintegrin polypeptide activities.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 60/221,838, filed 28 Jul. 2000, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to polypeptides having homology to the human Metalloproteinase-Disintegrin polypeptide family, to polynucleotides encoding such polypeptides, and to methods of making and use thereof.

BACKGROUND

[0003] The Metalloproteinase-Disintegrin polypeptides, also referred to herein as ADAM (“A Disintegrin And Metalloproteinase domain”) polypeptides or “ADAMs,” are a related group of multi-domain, type I membrane polypeptides. Certain members of the ADAM family of polypeptides are highly expressed in some cell types including, for example, reproductive tissue or muscle cells. In addition, members of the ADAM family of polypeptides are generally constitutively expressed throughout development.

[0004] A number of ADAM genes have now been identified, including fertilin α and β (involved in the integrin mediated binding and fusion of egg and sperm; previously known as PH-30 α and β), epididymal apical protein I, cyritestin, MDC (a candidate for tumor suppressor in human breast cancer), meltrin-a (mediates fusion of myoblasts in the process of myotube formation), MS2 (a macrophage surface antigen), and metargidin. In addition, a new ADAM family gene, named ADAMTS-1, containing a disintegrin and metalloproteinase domain with thrombospondin (TSP) motifs, has been shown to be closely associated with various inflammatory processes, as well as development of cancer cachexia (Kuno, K. et al., J. Biol. Chem. 272:556-562 (1997)). A new member of ADAM in Drosophila, called the kuzbanian gene (“KUZ”), was found to be involved in Drosophila neurogenesis (Rooke, J. et al., Science 273:1227-1231 (August 1996)).

[0005] Typical ADAM family polypeptides are cell surface polypeptides that consist of pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like repeat, transmembrane and cytoplasmic domains. In some ADAMs the metalloproteinase domain is believed to be involved in protein processing functions such as release of growth factors, adhesion proteins, and inflammatory factors. The disintegrin domain may play a role in integrin-mediated cell adhesion (cell to cell and cell to matrix) interactions, such as platelet aggregation, migration of tumor cells or neutrophils, and angiogenesis. These activities of the ADAM family of polypeptides are most likely mediated rough interactions with the substrates of the metalloproteinase and with integrins, with the substrates of the metalloproteinase binding to the metalloproteinase catalytic domain and inters binding to the disintegrin domain of the ADAM family of polypeptides. Because of their suspected roles in mediation of protein processing functions such as release of growth factors, adhesion proteins, and inflammatory factors and cell adhesion, the ADAM family of polypeptides are suspected of being associated with inflammation, cancer, allergy, reproductive, and vascular conditions. Characteristics and activities of the ADAM polypeptide family are described further in Black, R. A. and White, J. M., 1998, Curr. Opin. in Cell Biol. 10:654-659; and in Schlondorff, J. and Blobel, C. P, 1999, J. Cell Sci. 112:3603-3617; which are incorporated by reference herein.

SUMMARY OF THE INVENTION

[0006] Provided herein for the first time are polypeptide sequences having homology to metalloproteinase-Disintegrin (MPD) polypeptides and to the ADAM (“A Disintegrin And Metalloproteinase”) polypeptide family and functional domains contained in the ADAM family of polypeptides as well as methods of making and methods of use thereof.

[0007] The present invention provides a substantially purified polypeptide comprising a sequence selected from the group consisting of: SEQ ID Nos: 34, and 6-27; fragments of SEQ ID NO:6 having disintegrin activity; fragments of SEQ ID NO:8 having disintegrin activity; fragments of SEQ ID NO: 14 having disintegrin activity; fragments of SEQ ID NO:24 having metalloproteinase activity; fragments of SEQ ID NO:24 having disintegrin activity; SEQ ID NO:6 from about residue 43 to 148; SEQ ID NO:8 from about residue 1 to 366; SEQ ID NO:8 from about residue 38 to 366; SEQ ID NO: 14 from about residue 1 to 622; SEQ ID NO: 14 from about residue 84 to 622; SEQ ID NO: 14 from about residue 299 to 622; SEQ ID NO:21 from about residue 1 to 701; SEQ ID NO:24 from about residue 1 to 277; SEQ ID NO:24 from about residue 278 to 435; SEQ ID NO:25 from about residue 1 to 332; SEQ ID NO:25 from about residue 1 to 627; SEQ ID NO:26 from about residue 1 to 215; SEQ ID NO:26 from about residue 118 to 215; SEQ ID NO:26 from about residue 224 to 383; and SEQ ID NO:27 from about residue 29 to 574.

[0008] The invention also provides a substantially purified polypeptide comprising a sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 97% homologous to a sequence as set forth in SEQ ID NO:3-4, 6-11, 13-18, 20, or 23-27 wherein the polypeptide has a metalloproteinase or disintegrin activity.

[0009] The invention further provides a polypeptide, as set forth above, linked to a second polypeptide, wherein the second polypeptide is a leucine zipper polypeptide, an Fc polypeptide, or a peptide linker moiety.

[0010] The invention further includes an isolated polynucleotide encoding a polypeptide of as set forth above, as well as vectors comprising the polynucleotide, and recombinant host cells comprising a polynucleotide of the invention.

[0011] The invention also provides a method for producing a polypeptide, comprising culturing a host cell containing a polynucleotide of the invention under conditions promoting expression of the polypeptide.

[0012] The invention further provides a polypeptide produced by culturing a host cell comprising a polynucleotide of the invention under conditions to promote expression of the polypeptide.

[0013] The invention provides a substantially purified antibody that specifically binds to a polypeptide of as set forth above. In one embodiment, the the antibody is a monoclonal antibody. In another embodiment, the antibody is a human or humanized antibody.

[0014] Also provided by the invention is a method of designing an inhibitor or binding agent of a polypeptide as set forth above or herein, comprising determining the three-dimensional structure of the polypeptide, analyzing the three-dimensional structure for binding sites of substrates or ligands, designing a molecule that is predicted to interact with the polypeptide, and determining the inhibitory or binding activity of the molecule.

[0015] The invention further provides a method for identifying an agent that modulates an activity of a polypeptide as set forth herein, comprising contacting the agent with the polypeptide under conditions such that the agent and the polypeptide interact; and determining activity of the polypeptide in the presence of the agent compared to a control (e.g., the polypeptide in the absence of the agent) wherein a change in activity is indicative of an agent that modulates the polypeptide's activity. In one embodiment, the activity of the polypeptide is selected from the group consisting of disintegrin activity, cell adhesion activity, angiogenic activity, metalloproteinase activity, and a combination thereof (e.g., metalloproteinase and disintegrin activity). In another embodiment, the agent is selected from the group consisting of an antibody, a small molecule, a peptide, and a peptidomimetic.

[0016] The invention also provides a method for modulating angiogenesis in a cell or mammal, comprising contacting or administering to the cell or mammal a polypeptide of the invention as set forth herein in an amount effective to modulate disintegrin activity. The cell may be contacted in vitro or in vivo.

[0017] Also provided by the invention is a method for modulating endothelial cell migration, comprising contacting the endothelial cell with a polypeptide of the invention, as set forth herein.

[0018] The invention provides a method of inhibiting the binding of an integrin to a ligand comprising contacting or administering to a cell or mammal that expresses the integrin an effective amount of a polypeptide having disintegrin activity. In one embodiment, the mammal is afflicted with a condition selected from the group consisting of ocular disorders; malignant and metastatic conditions; inflammatory diseases; osteoporosis and other conditions mediated by accelerated bone resorption; restenosis; inappropriate platelet activation, recruitment, or aggregation; thrombosis; and a condition requiring tissue repair or wound healing.

[0019] The polypeptides of the invention and the methods of the invention include polypeptides in the form of a multimer (e.g., a dimer or trimer). The multimer can comprise an Fc polypeptide, a leucine zipper, or a peptide linker.

[0020] The invention further provides a system for analyzing polypeptides or polynucleotides of the invention comprising a data set representing a set of one or more polypeptides as set forth herein; a computer; and a computer algorithm in an executable format on the computer for analyzing the polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows polypeptide sequences of the invention in single letter amino acid code. “X” represents any amino acids or a plurality of any amino acids.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The invention provides polypeptides having or predicted to have metalloproteinase and/or disintegrin activity. These metalloroteinase-like/disintegrin-like (MPD) polypeptides find use in the treatment and diagnosis of integrin-associated and metalloproteinase-associated diseases and disorders and well as use in the development of diagnositics and related therapeutics.

[0023] Matrix metalloproteinases (MMPs) compose a family of structurally similar zinc-dependent enzymes that degrade all of the major components of the extracellular matrix and play a major role in tissue remodeling and repair associated with development and inflammation (Matresian, Trends Genet. 6:121-125, 1990; and Woessner, FASEB J. 5:2145-2154, 1991). MMPs include the collagenases, gelatinases A and B, the stromelysins, matrilysin, metalloelastase, and the membrane-type matrix metalloproteinases. Over-expression and activation of MMPs have been linked with a range of diseases, such as arthritis, cancer, and multiple sclerosis. MMPs classically have been implicated in basement membrane destruction associated with late-stage tumor cell invasion and metastasis, one MPD member, matrilysin, has been shown to be expressed in early stage human colorectal tumors. (Wilson et al., Proc. Natl. Acad. Sci. USA 94:1402-1407, 1997). Abnormal expression of MMPs can contribute to destructive processes including tumor invasiveness (Mignatti and Rifkin, Cell 47:487498, 1986; and Khokha et al., Science 243:947-950, 1989), arthritis (Dean et al., J. Clin. Invest. 84:678-685, 1989; and McCachren, Arthritis Rheumn 34:1085-1093, 1991), and atherosclerosis (Henney et al., Proc. Natl. Acad. Sci. USA, 88:8154-8158, 1991). The metalloproteinases identified herein are candidate proteins contributing to the pathogenesis of, for example, inflammatory diseases and disorders. Accordingly, the metalloproteinase polypeptides provided herein can be used to treat inflammatory disorders and as a source for the development of inhibitors both in silico and in vitro. Inhibitors to the metalloproteinases provided herein can ameliorate diseases and disorders associated with excessive proteinase activity including, for example, inflammation.

[0024] The disintegrin domain of ADAM family proteins functions in the prevention of integrinmediated cell to cell and cell to matrix interactions, such as platelet aggregation, adhesion, and migration of tumor cells or neutrophils, and angiogenesis. Previously described disintegrins, such as contortrostatin (Trikha et al., Cancer Research 54:4993-4998, 1994) have been used to inhibit human metastatic melanoma (M24 cells) cell adhesion to type I collagen, vitronectin, and fibronection, but not laminin. Further, contortrostatin inhibits lung colonization of M24 cells in a murine metastasis model.

[0025] The structure of most metalloproteinase-disintegrin polypeptides includes a signal domain, a metalloproteinase domain, a disintegrin domain, a transmembrane domain and a cytoplasmic domain. For example, the typical structural elements common to various members of the ADAM family of polypeptides include, in N-to-C order, a signal sequence, a prodomain, a metalloproteinase domain, a disintegrin domain, a cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain. There are certain key residues within the metalloproteinase domains/motifs (e.g., the HExGHxxGxxHD motif (SEQ ID NO:28)) such that substitutions of those extremely conserved residues are likely to be associated with an altered function or lack of function for the polypeptide. ADAMs with the conserved metalloprotease active site sequence of SEQ ID NO:28 include ADAMs 1, 8-10, 12-13, 15-17, 19-21, 24-26, 28, and 30. The metalloproteinase catalytic domains also contain four conserved cysteines that may be required for the formation of a functional polypeptide structure through disulfide bonds. There are 31 highly conserved cysteines in the disintegrin and cysteine rich region; almost all of the ADAM family of polypeptides have these 31 cysteines. The skilled artisan will recognize that the boundaries of these regions within the polypeptides are approximate and that the precise boundaries of such domains (which can be predicted by using computer programs available for that purpose) can differ from member to member within the ADAM family of polypeptides.

[0026] The ADAM family of polypeptides is reasonably well conserved, with the human family members similar to each other and to ADAM family members from other species such as mouse, rat, and even Drosophila inelaiogaster and Caenorhlabditis elegans (see, e.g., Yamamoto et al., Immunol. Today, 20(6):278, 1999; and the following Internet websites for more information (www): gene.ucl.ac.uktusers/hester/metalo.html; uta.fi/˜loiika/ADAMs/HADAMs.htmnl; and people.Virginia.EDU/˜jag6n/Table_of_the_ADAMs.html). However, subfamilies of the ADAM family of polypeptides can be defined on the basis of sequence similarity and related biological activities. One such subfamily comprises the ADAM10 and ADAM17/TACE polypeptides, which show greater sequence similarity to each other compared to other members identified so far within the ADAM family of polypeptides. ADAMs 10 and 17 have 21 cysteines in the disintegrin-cysteine rich region in contrast to the 31 conserved cysteines in this region among the other ADAMs. Accordingly, ADAMs may have from 20 to 31 conserved cysteines (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 conserved cysteines). ADAM17/TACE and ADAM-10 are also “sheddases,” meaning they are believed to cleave and release the extracellular domains of other membrane proteins. The major function of another subfamily may be to bind integrins or other proteins. ADAM-2, for example, is processed to remove both the prodomain and the metalloproteinase catalytic domain so as to expose the disintegrin domain and allow it to bind to its cognate. Another subfamily that can be defined are the ADAMs that appear to be testis-specific; these proteins include ADAMs 2-3, 16, 18, 20-21, 24-26, and 29-30; with ADAMs 5 and 6 being primarily testis-specific.

[0027] Polypeptides of the ADAM family are expressed in many cell types including, for example, uritogenital tissues (e.g., kidney tissue and reproductive tissue), neurologic tissue, and muscle cells. Some binding partners for ADAM polypeptides are expressed, for example, by endothelial cells and T cells, as displayed by the disintegrin-cysteine rich domains of several ADAM family polypeptides binding to endothelial cells, at least partly through interaction with integrins, and to T cells. The interactions between members of the ADAM family of polypeptides and their binding partners are likely involved in mediating interactions between cell types including reproductive tissue, neurologic tissue, and muscle cells, and binding-partner-expressing endothelial cells and T cells.

[0028] The disintegrin domain of some ADAM family polypeptides can interact with binding partners such as cell surface integrins (see, e.g., co-pending International Application Serial No. PCT/US01/05701, the disclosure of which is incorporated herein by reference in its entirety). By binding to one or more binding partners, a disintegrin domain polypeptide can inhibit the biological activities (e.g., angiogenesis) mediated via binding of an ADAM polypeptide to its binding partner. Because some ADAM family polypeptides exhibit integrin-binding activities via the disintegrin domain, modulation of disintegrin activity will modulate adhesion, e.g., the role of ADAMs 1 and 2 in sperm binding to egg and the role of ADAM-9 in interactions of glomerular and tubular epithelial cells with the basal laminae in renal tissue. The degree to which individual members of the ADAM family of polypeptides and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay methods, such as inhibition of endothelial cell migration by disintegrin-Fc constructs, and the like. Particularly suitable assays to detect or measure the binding between ADAM polypeptides and their binding partners are FACS analyses. Additional assays for evaluating the biological activities and partner-binding properties of ADAM family polypeptides are described below. Polypeptides of the invention lacking a metalloproteinase domain are contemplated by the present invention and may act as a dominant negative with respect to the metalloproteinase activity of other ADAM family polypeptides (see, e.g., International Publication WO/______, entitled, “A HUMAN DISINTEGRIN PROTEIN,” filed 27 Jul. 2001, the disclosure of which is incorporated herein by reference in its entirety).

[0029] Polypeptides of the ADAM family are involved in inflammation, cancer, allergy, reproductive, neural disorders and diseases, angiogenesis and vascular diseases or conditions that share as a common feature integrin-associated interactions via disintegrin activity and/or protein degredation via metalloproteinase activity. Examples of inflammation, cancer, allergy, reproductive, neural disorders and diseases, angiogenesis and vascular conditions that are known or are likely to involve the biological activities of ADAM polypeptides are rheumatoid arthritis, septic shock, glomerular diseases, acute renal failure, Alzheimer's disease, and inappropriate bone resorption. Blocking or inhibiting the interactions between members of the ADAM family of polypeptides and their substrates, ligands, receptors, binding partners, or other interacting polypeptides is an aspect of the invention and provides methods for treating, modulating, or ameliorating these diseases and conditions through the use of inhibitors or modulators of ADAM polypeptide activity. In one embodiment, interaction between members of the ADAM family of polypeptides and their cognates is affected by contacting a sample containing an ADAM family polypeptide or its cognate with an MPD polypeptide or anti-MPD antibody.

[0030] For certain conditions involving too little disintegrin activity, methods of treating or ameliorating these conditions comprise increasing the amount or activity of, for example, MPD polypeptides having disintegrin activity by providing such polypeptides or active fragments or fusion polypeptides thereof, or by providing agents that activate endogenous or exogenous MPD polypeptides. Additional uses for MPD polypeptide include diagnostic reagents for inflammation, cancer, allergy, reproductive, neural disorders, and vascular diseases; research reagents for investigation of integrin polypeptides and fertilization processes, purification and processing of integrins and/or endothelial cells or T cells; or as a carrier/targeting polypeptide to deliver therapeutic agents to cells.

[0031] As used herein, both “protein” and “polypeptide” mean any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation), and include natural proteins, synthetic or recombinant polypeptides and peptides as well as a recombinant molecule consisting of a hybrid with one portion, for example, comprising all or part of an MPD amino acid sequence and a second portion being encoded by all or part of a different nucleotide sequence. Typically the protein or polypeptide is substantially pure of other components from which it is normally present in nature. The term “substantially pure” or “purified” when referring to a polypeptide, means a polypeptide that is at least 30% free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the substantially pure polypeptide of the invention is at least 35 to 50%; preferably 60 to 70%; more preferably at least 75% to 90%; and most preferably at least 99% by weight purified from other naturally occurring molecules. A substantially pure polypeptide of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant polynucleotide encoding the polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, e.g., chromatography, PAGE, or HPLC analysis.

[0032] As used herein an “MPD polypeptide” means a polypeptide that contains or comprises an amino acid sequence as set forth in FIG. 1; polypeptides having substantial homology or substantial identity to the sequences set forth in FIG. 1; fragments of the foregoing sequences; and conservative variants of the foregoing. The invention provides MPD polypeptides comprising a sequence as set forth in SEQ ID Nos:1 to 27.

[0033] MPD polypeptides comprising sequence associated with disintegrin activity include SEQ ID NO:6; SEQ ID NO:6 from about residue 43 to 148; SEQ ID NO:8 from about residue 1 to 366; SEQ ID NO:8 from about residue 38 to 366; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:14 from about residue 1 to 622; SEQ ID NO: 14 from about residue 84 to 622; SEQ ID NO: 14 from about residue 299 to 622; SEQ ID NO: 16; SEQ ID NO:21 from about residue 1 to 701; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:24 from about residue 278 to 435; SEQ ID NO:25 from about residue 1 to 627; SEQ ID NO:26; and SEQ ID NO:26 from about residue 224 to 383, termed herein “MPD disintegrin polypeptides,” (MPDdis). A used herein, the term “between about” or “from about” will be understood to include sequences between any such referenced residues of a sequence. For example, “from about 43 to 148” means 44 to 148, 45 to 148, 46 to 148, and so on; and 43 to 147, 43 to 146, and so on.

[0034] MPD polypeptides comprising sequences associated within metalloproteinase activity include SEQ ID NO: 14 from about residue 1 to 622; SEQ ID NO: 14 from about residue 84 to 622; SEQ ID NO:18; SEQ ID NO:21 from about residue 1 to 701; SEQ ID NO:22; SEQ ID NO:24; SEQ I) NO:24 from about residue 1 to 277; SEQ ID NO:25 from about residue 1 to 332; SEQ ID NO:25 from about residue 1 to 627; SEQ ID NO:26 from about residue 1 to 215; SEQ ID NO:26 from about residue 118 to 215; and SEQ I) NO:26, termed herein “MPD metalloproteinase polypeptides.”

[0035] The MPD polypeptides have been shown to have a high degree of homology to members of the ADAM family and related metalloproteinase/disintegrin polypeptides and thus have a predicted function or activity of an ADAM polypeptide, a disintegrin polypeptide, or a metalloproteinase polypeptide. Accordingly, the invention provides an MPD polypeptide comprising a sequence selected from the group consisting of SEQ ID Nos: 1 to 26, and 27. In one embodiment, the MPD polypeptide has disintegrin activity, metalloproteinase activity, or a combination thereof. Methods of determining whether a polypeptide of the invention has a desired disintegrin activity or metalloproteinase activity can be accomplished by assaying the polypeptide by any of the methods described herein below.

[0036] A number of conserved sequences have been identified in ADAM and matrix metalloproteinases (MMPs) including, for example, the HExGHxxGxxHD motif (SEQ ID NO:28). In addition, a potential conserved motif includes a LNlx(YV)(AN)LVGLE(V/I)WT motif (SEQ ID NO:29). For example, SEQ ID Nos:4 to 5, 10, 14, 21 to 22, and 25 to 26 comprise the sequence HexGHxxGxxHD (SEQ ID NO:28) at residues 208 to 219, 15 to 26, 229 to 240, 555 to 566, 3 to 14, 269 to 280, and 154 to 165, respectively. SEQ ID NO:24 comprises a sequence that has substantial identity to the conserved HexGHxxGxxHD motif. Thus, a polypeptide comprising SEQ ID NO: 4, 5, 10, 14, 21, 22, 24, 25, or 26 is predicted to have metalloproteinase activity. SEQ ID Nos:4, 8, 10, 14, 18 to 19, 21, and 25 comprise a sequence having identity with the LNIx(WV)(A/V)LVGLE(V/I)WT motif and thus a polypeptide comprising SEQ ID NO: 4, 8, 10, 14, 18 to 19, 21, or 25 is predicted to have metalloproteinase activity. The invention also provides SEQ ID Nos:2, 3, 7, 9, 17, 20, and 27 which have a high degree of homology to the Testicular Metalloproteinase-like, Disintegrin-like, Cysteine rich (TMDC) protein family, including TMDC III, TMDC IVA, and TMDC IVC. Table 1 shows the relative identity of representative polypeptides of the invention with TMDC protein family members. Accordingly, polypeptides comprising sequences as set forth in SEQ ID Nos: 2, 3, 7, 9, 17, 20, or 27, and fragments thereof having metalloproteinase activity and/or disintegrin activity are provided herein. TABLE 1 SEQUENCE Homology TMDC family member SEQ ID NO: 2 99% TMDCIII SEQ ID NO: 3 91% TMDCIVC SEQ ID NO: 7 85% TMDCIVA SEQ ID NO: 9 48% TMDCIVA SEQ ID NO: 17 53% TMDCIVA SEQ ID NO: 20 90% TMDCIV SEQ ID NO: 27 89% TMDCIVA

[0037] In addition, to the sequences above having homology to TMDC family members, the invention also provides polypeptides having homology to the ADAM (A Disintegrin And Metaloproteinase) family of proteins. Such polypeptides include metaloproteinase domains. Preferably the metalloproteinase domain of the polypeptides comprising SEQ ID Nos:4, 10, 14, 21, 25, and 26 comprise a sequence from about amino acids residues 65 to 274 of SEQ ID NO:4; 24 to 235 of SEQ ID NO: 10; 85 to 290 of SEQ ID NO: 14; 202 to 411 of SEQ ID NO:21; 123 to 332 of SEQ ID NO:25; and 118 to 215 of SEQ ID NO:26. One of skill in the art will recognize that the N-terminal and C-terminal residues of the respective metalloproteinase domains are approximate and variations of about 1 to 10 amino acid(s) from either end of the domain will not depart from the scope of the present invention. For example, the addition of amino acids to either end of the domain may not change the molecule's activity. The effects of any such modification can be assayed using the methods described herein. Polypeptides comprising sequences as set forth in SEQ ID Nos: 2 to 5, 7 to 10, 14, 17 to 19, 21 to 22, and 24 to 27 may also have disintegrin activity in addition to metalloproteinase activity.

[0038] The disintegrin domain is typically characterized as containing a conserved motif having a sequence CGN(G/K)x(LN)(E/D)x(G/N)EECDCG (SEQ ID NO:30) (herein after the “CGN-GEEC” motif). The present invention provides polypeptides having disintegrin activity characterized as having a motif with substantial identity to the CGN(G/K)×(LIV)(E/D)×(G/N)EECDCG (SEQ ID NO:30). For example, SEQ ID Nos:4, 10, 14, 21, and 24 to 26 contain the CGN-GEEC motif and thus a polypeptide comprising SEQ ID NO: 4, 10, 14, 21, 24, 25, or 26 is predicted to have disintegrin activity. SEQ ID NO: 11 has a putative CGN-GEEC sequence at residues 43 to 57 and thus a polypeptide comprising SEQ ID NO: 11 is predicted to have disintegrin activity. In addition, ADAM family of proteins are characterized as having a number of conserved cysteine residues in their disintegrin and cysteine-rich domains. For example, SEQ ID Nos:6, 8, 12, 13, 16, and 23, when aligned with a number of ADAM family members (e.g., ADAM9 (accession no. NP 003807, which is incorporated herein by reference)), align with the conserved cysteine residues in the disintegrin domain of such ADAM family members. Thus, the invention also provides polypeptides comprising a sequence as set forth in SEQ ID Nos:6, 8, 12, 13, 16, and 23 having disintegrin activity.

[0039] Table 2 provides a summary of the relative domains and residues characterizing the domains of some of the polypeptides of the invention. The relative domains and residues corresponding to such activity are estimates based on similar molecules and computer algorithms and accordingly may vary slightly depending upon a number of factors including, for example, the source of material, the cell type used, the expression system used, and the like. Such factors will be apparent and appreciated by one of skill in the art. TABLE 2 Predicted Predicted Predicted Disintegrin Predicted Cytoplasmic Metalloproteinase domain Transmembrane domain Sequence domain comprises comprises domain comprises comprises (SEQ ID NO:) residues: residues: residues: residues: 4 65 to 274 283 to 564 565 to 585 586 to 686 5 1 to 46 6 1 or 43 to 148 8 1 or 38 to 366 10 1, 24 or 118 to 235 244 to 528 529 to 539 540 to 751 11 1 to 126 12 1 to 121 13 1 to 66 14 85 to 290 299 to 622 623 to 642 643 to 660 16 1 to 60 18 1 to 56 19 1 to 56 21 30 or 202 to 411 420 to 710 711 to 725 726 to 812 22 1 to 38 24 1 to 277 278 to 435 25 123 to 332 343 to 627 628 to 645 646 to 811 26 118 to 215 224 to 383

[0040] The invention further provides polypeptides having metalloproteinase activity and/or disintegrin activity comprising SEQ ID NO: 15. For example, SEQ ID NO: 15 has homology to ADAM 9 immediately following the metalloproteinase and before the disintegrin domain of ADAM 9.

[0041] A polypeptide of the invention also encompasses an amino acid sequence that has a sufficient or a substantial degree of identity or similarity to a sequence set forth in FIG. 1. Substantially identical sequences can be identified by those of skill in the art as having structural domains and/or having biological activity in common with an MPD polypeptide. Methods of determining similarity or identity may employ computer algorithms such as, e.g., BLAST, FASTA, and the like.

[0042] The phrase “substantially identical,” in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 50%, 60%, preferably 80% or 85%, more preferably 90 to 95%, and most preferably 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured by, for example, a sequence comparison algorithm or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementary when the test sequence has substantial identity to a reference sequence. A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 1800, usually about 50 to 200, more usually about 70 to 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0043] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0044] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFlT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.

[0045] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987), and is similar to the method described by Higgins & Sharp, CABIOS 5:151 (1989). The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

[0046] Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, as described in Altschul et al., J. Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www-ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy a positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (I(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0047] Alternatively, the percent identity of two amino acid or two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0048] One of skill will recognize that individual substitutions, deletions or additions to a nucleic acid sequence, peptide, or polypeptide sequence that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in a molecule having substantially the same biological activity (e.g., disintegrin and/or metalloproteinase activity). For example, an alteration that results in the substitution of an amino acid with a chemically similar amino acid is a conservatively modified variant. Conservative substitution tables providing functionally similar amino acids are known in the art. The following six groups each contain amino acids that are conservative substitutions for one another 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see, e.g., Creighton, Proteins (1984)).

[0049] One indication that two polynucleotides or polypeptides are substantially identical is that the polypeptide encoded by a first polynucleotide is immunologically cross reactive with the antibodies raised against the polypeptide encoded by a second polynucleotide. Another indication that two polynucleotides are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions.

[0050] Polypeptides derived from the MPD polypeptides of the invention by any type of alteration (e.g., insertions, deletions, or substitutions of amino acids; changes in the state of glycosylation of the polypeptide; refolding or isomerization to change its three-dimensional structure or self-association state; and changes to its association with other polypeptides or molecules) are also encompassed by the invention. Therefore, the polypeptides provided by the invention include polypeptides characterized by amino acid sequences similar to those as set forth in FIG. 1, but into which modifications are naturally provided or deliberately engineered. A polypeptide that shares biological activities in common with a polypeptide comprising a sequence as set forth in SEQ ID NO:1-26, or 27 having disintegrin activity and/or metalloproteinase activity are encompassed by the invention.

[0051] The present invention encompasses the use of various forms of MPD disintegrin polypeptides or domains that retain at least one activity selected from the group consisting of integrin binding activity, inhibition of endothelial cell migration, and inhibition of angiogenesis. A MPD disintegrin polypeptide/domain (MPDdis) is intended to encompass polypeptides comprising all or part of an MPD polypeptide of the invention having disintegrin activity. In a preferred embodiment, an MPDdis contains all or part of an MPD disintegrin domain, with or without other domains (such as the cysteine-rich region), as well as related forms including, but not limited to: (a) fragments, (b) variants, (c) derivatives, (d) fusion polypeptides, and (e) multimeric forms (multimers). The ability of these related forms to inhibit integrin binding, endothelial cell migration, and/or inhibition of angiogenesis may be determined in vitro or in vivo by using methods such as those exemplified below or by using other assays known in the art.

[0052] One of skill in the art can easily assay for activity using the methods described herein. Such methods measure, for example, metalloproteinase activity, disintegrin activity, or a biological activities exhibited by members of the ADAM family of polypeptides including, without limitation, cell adhesion. For example, anti-MPD antibodies, which neutralize MPD activity (e.g, metalloproteinase activity and/or disintegrin activity) can be used to assay for similar polypeptides by contacting an anti-MPD antibody with a polypeptide of interest and determining if the activity associated with the polypeptide of interest is neutralized. In addition, the cross-reactivity of an antibody that specifically binds to an MPD polypeptide of the invention with another polypeptide of interest is indicative that the polypeptide of interest shares structural characteristics (e.g., primary, secondary, or tertiary protein characteristics) with an MPD polypeptide of the invention.

[0053] The invention provides both full length and mature forms of MPD polypeptides. Full-length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated. The amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame (“ORF”) of a cDNA molecule. Several full-length polypeptides may be encoded by a single genetic locus if multiple mRNA forms are produced from that locus by alternative splicing or by the use of multiple translation initiation sites. The “mature form” of a polypeptide refers to a polypeptide that has undergone post-translational processing steps, if any, such as, for example, cleavage of the signal sequence or proteolytic cleavage to remove a prodomain. Multiple mature forms of a particular full-length polypeptide may be produced, for example, by imprecise cleavage of the signal sequence, or by differential regulation of proteases that cleave the polypeptide. The mature form(s) of such polypeptide may be obtained by expression, in a suitable mammalian cell or other host cell, of a polynucleotide that encodes the full-length polypeptide. The sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites. The MPD polypeptides of the invention also include polypeptides that result from post-transcriptional or post-translational processing events such as alternate mRNA processing which can yield a truncated but biologically active polypeptide, for example, a naturally occurring soluble form of the polypeptide. Also encompassed within the invention are variations attributable to proteolysis such as differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptide (generally from 1-5 terminal amino acids).

[0054] A polypeptide of the invention may be prepared by culturing transformed or recombinant host cells under culture conditions suitable to express a polypeptide of the invention. The resulting expressed polypeptide may then be purified from such culture using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the polypeptide may also include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®D; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, a polypeptide of the invention may also be expressed in a form that will facilitate purification. For example, it may be expressed as a fusion polypeptide, joined to, for example, maltose binding polypeptide (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.), and InVitrogen, respectively. The polypeptide can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (“Flag”) is commercially available from Kodak (New Haven, Conn.). Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous recombinant polypeptide. The polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the invention as a “substantially purified polypeptide”; such purified polypeptides include antibodies that specifically bind to an MPD polypeptide, fragment, variant, and the like. A polypeptide of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a polynucleotide encoding a polypeptide of the invention.

[0055] It is also possible to utilize an affinity column such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention. In this aspect of the invention, proteins that bind a polypeptide of the invention (e.g., an anti-MPD antibody of the invention) can be bound to a solid phase support or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface. Adherence of, for example, an anti-MPD antibody of the invention to a solid phase surface can be accomplished by any means, for example, magnetic microspheres can be coated with these polypeptide-binding proteins and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding proteins thereon. Anti-NWD antibodies bind cells having polypeptides of the invention on their surface (e.g., an extracellular domain of MPD). Unbound cells (e.g., cell lacking and MPD polypeptide) are washed away from the bound cells. This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution. Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are preferably directed to cleaving the cell-surface binding partner. Alternatively, mixtures of cells suspected of containing polypeptide-expressing cells of the invention are first incubated with a biotinylated binding polypeptide of the invention. Incubation periods are typically at least one hour in duration to ensure sufficient binding to polypeptides of the invention. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the cells to the beads. Use of avidin-coated beads is known in the art (see, Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.

[0056] A polypeptide of the invention may also be produced by known conventional chemical synthesis. Methods for constructing the polypeptides of the invention by synthetic means are known to those skilled in the art. The synthetically-constructed polypeptide sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with a native polypeptides may possess biological properties in common therewith, including biological activity. Thus, the synthesized polypeptides may be employed as biologically active or immunological substitutes for natural, purified polypeptides in screening of therapeutic compounds, and in immunological processes for the development of antibodies.

[0057] The desired degree of purity depends on the intended use of the polypeptide. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.

[0058] Species homologues of MPD polypeptides and polynucleotides encoding the polypeptides are also provided by the invention. As used herein, a “species homologue” is a polypeptide or polynucleotide with a different species of origin from that of a given polypeptide or polynucleotide, but with significant sequence similarity to the given polypeptide or polynucleotide. Species homologues may be isolated and identified by making suitable probes or primers from polynucleotides encoding the polypeptides provided herein and screening a suitable nucleic acid source from the desired species. Alternatively, homologues may be identified by screening a genome database containing sequences from one or more species utilizing a sequence (e.g., nucleic acid or amino acid sequence) of an MPD of the invention. Such genome databases are readily available for a number of species (e.g., on the world wide web (www) at tigr.org/tdb; genetics.wisc.edu; stanford.edu/-ball; hiv-web.lanl.gov; ncbi.nlm.nig.gov; ebi.ac.uk; and pasteur.fr/other/biology). The invention also encompasses allelic variants of MPD polypeptides and nucleic acids encoding them that are naturally-occurring alternative forms of such polypeptides and polynucleotides in which differences in amino acid or nucleotide sequence are attributable to genetic polymorphism.

[0059] Intermediate Sequence Search (ISS) can be used to identify closely related as well as distant homologs by connecting two proteins through one or more intermediate sequences. ISS repetitively uses the results of the previous query as new search seeds. Saturated BLAST is a package that performs ISS. Starting with a protein sequence, Saturated BLAST runs a BLAST search and identifies representative sequences for the next generation of searches. The procedure is run until convergence or until some predefined criteria are met. Saturated BLAST is available on the world wide web (www) at: bioinformatics.burnham-inst.org/xblast (see also, Li et al. Bioinformatics 16(12): 1105, 2000).

[0060] Fragments of the MPD polypeptides of the invention are encompassed by the invention and may be in linear form or cyclized using known methods (see, e.g., H. U. Saragovi, et al., BiolTechnology 10, 773 (1992); and R. S. McDowell, et al., J. Amer. Chem. Soc. 114:9245 (1992), both of which are incorporated by reference herein). Peptide fragments of MPD polypeptides of the invention, and polynucleotides encoding such fragments include amino acid or nucleotide sequence lengths that are at least 25% (more preferably at least 50%, 60%, or 70%, and most preferably at least 80%) of the length of an MPD polypeptide or polynucleotide. Preferably such sequences will have at least 60% sequence identity (more preferably at least 70%-75%, 80%-85%, 90%-95%, at least 97%-97.5%, or at least 99%, and most preferably at least 99.5%) with an MPD polypeptide or polynucleotide when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the invention are polypeptides, peptide fragments, and polynucleotides encoding such fragments, that contain or encode a segment preferably comprising at least 8 to 10, or more preferably at least 20, or still more preferably at least 30, or most preferably at least 40 contiguous amino acids. Such polypeptides and fragments may also contain a segment that shares at least 70% (at least 75%, 80%-85%, 90%-95%, at least 97%-97.5%, or at least 99%, and most preferably at least 99.5%) with any such segment of, for example, any of the ADAM family polypeptides, when aligned so as to maximize overlap and identity while minimizing sequence gaps. Visual inspection, mathematical calculation, or computer algorithms can determine the percent identity.

[0061] The invention also provides soluble forms of MPD polypeptides comprising certain fragments or domains of these polypeptides. Soluble fragments having disintegrin activity are of particular interest. For example, an amino acid sequence beginning with a highly conserved CGN-GEEC sequence (as discussed above) but which lacks a transmembrane region (see, e.g., Table 2). Transmembrane regions can be identified using publicly available computer algorithms. Other soluble forms include polypeptides comprising SEQ ID NO:6 beginning at an amino acid between and including residues 1 and 43 to 148; SEQ ID NO:8 beginning at an amino acid between and including residues 1 and 38 to 366; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO: 14 beginning at an amino acid between and including residues 1 and 84 to 622; SEQ ID NO: 14 beginning at an amino acid between and including residues 1 and 299 to 622; SEQ ID NO:16; SEQ ID NO:21 from about residue 1 to 701; SEQ ID NO:23; SEQ ID NO:24 beginning at an amino acid between and including residues 1 and 278 to 435; SEQ ID NO:25 from about residue 1 to 627; and SEQ ID NO:26 beginning at an amino acid between and including residues 1 and 224 to 383. In such polypeptides can be secreted from the cell in which it is expressed. The intracellular and transmembrane domains of polypeptides of the invention can be identified in accordance with known techniques for determination of such domains from sequence information. For example, alignment of the polypeptide sequences of the invention with other members of the ADAM family of polypeptides having known domains will provide information regarding the domains of the polypeptides of the invention. One of skill in the art will recognize that slight modifications in the range of sequences of a particular domain can be made without affecting the molecule's biological activity. Accordingly, changes in the identified sequences of 1, 2, 3, 4, or 5 to 10 amino acids in either direction of the particular domain are encompassed by the present invention.

[0062] In another aspect of the invention, a polypeptide may comprise various combinations of ADAM polypeptide domains, such as a metalloproteinase domain, a disintegrin domain, or a cytoplasmic domain. Accordingly, polypeptides of the invention and polynucleotides include those comprising or encoding two or more copies of a domain such as the metalloproteinase domain, two or more copies of a domain such as the disintegrin domain, or at least one copy of each domain, and these domains may be presented in any order within such polypeptides. Also included are recombinant polypeptides and the polynucleotides encoding the polypeptides wherein the recombinant polypeptides are “chimeric polypeptides” or “fusion polypeptides” and comprise an MPD sequence as set forth in SEQ ID NO: 1-26 or 27 operatively linked to a second polypeptide. The second polypeptide can be any polypeptide of interest having an activity or function independent of, or related to, the function of an MPD polypeptide. For example, the second polypeptide can be a domain of a related but distinct member of the ADAM family of polypeptides such as, for example, an extracellular, cytoplasmic, metalloprotease, or transmembrane domain of an ADAM polypeptide. The term “operatively linked” is intended to indicate that the MPD sequence and the second polypeptide sequence are fused in-frame to each other. The second polypeptide can be fused to the N-terminus or C-terminus of an MPD sequence as set forth in FIG. 1. For example, in one embodiment the fusion polypeptide is a GST-MPD fusion polypeptide in which an MPD sequence is fused to the C-terminus of the GST sequences. Such fusion polypeptides can facilitate the purification of recombinant MPD sequences. In another embodiment, the fusion polypeptide is an MPD sequence comprising a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of an MPD polypeptide can be increased through use of a heterologous signal sequence. As another example, an MPD polypeptide or fragment thereof may be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein, or to a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells. Further, fusion polypeptides can comprise, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG® peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant polypeptide. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG® peptide in the presence of certain divalent metal cations, as described in U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11 hybridoma cell line has been deposited with the ATCC under accession no. HB9259. Monoclonal antibodies that bind the FLAG® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Conn.

[0063] Encompassed by the invention are oligomers or fusion polypeptides that comprise an MPD polypeptide. Oligomers that can be used as fusion partners can be in the form of covalently linked or non-covalently4inked multimers, including dimners, trimers, or higher oligomers. In one aspect of the invention, the oligomers maintain the binding ability or catalytic ability of the polypeptide components and provide therefor, bivalent, trivalent, and the like, binding or catalytic sites. In an alternative embodiment the invention is directed to oligomers comprising multiple polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides. Such peptides can be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below.

[0064] Typically a linker will be a peptide linker moiety. The length of the linker moiety is chosen to optimize the biological activity of the polypeptide comprising an MPD sequence and can be determined empirically without undue experimentation. The linker moiety should be long enough and flexible enough to allow an MPD polypeptide to freely interact with a substrate or ligand. The preferred linker moiety is a peptide between about one and 30 amino acid residues in length, preferably between about two and 15 amino acid residues. Preferred linker moieties are —Gly—Gly—, GGGGS (SEQ ID NO:31), (GGGGS)N (SEQ ID NO:32), GKSSGSGSESKS (SEQ ID NO:33), GSTSGSGKSSEGKG (SEQ ID NO:34), GSTSGSGKSSEGSGSTKG (SEQ ID NO:35), GSTSGSGKPGSGEGSTKG (SEQ ID NO:36), or EGKSSGSGSESKEF (SEQ ID NO:37). Linking moieties are described, for example, in Huston, J. S., et al., PNAS 85:5879 (1988), Whitlow, M., et al., Protein Engineering 6:989 (1993), and Newton, D. L., et al., Biochemistry 35:545 (1996). Other suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated by reference. A DNA sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as, a DNA sequences encoding an MPD polypeptide or fragment thereof, using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding the linker can be ligated between the sequences. In particular embodiments, a fusion polypeptide comprises from two to four soluble MPD polypeptides, separated by peptide linkers.

[0065] In embodiments where variants of an MPD polypeptide are constructed to include a membrane-spanning domain, they will form a Type I membrane polypeptide. In such embodiments, it is preferable to link the fusion partner to the C-terminus of the MPD polypeptide. Alternatively, the membrane-spanning polypeptides can be fused with known extracellular receptor domain polypeptides, for which the ligand is also known. Such fusion polypeptides can then be manipulated to control the intracellular signaling pathways triggered by the bound MPD polypeptide. Polypeptides that span the cell membrane can also be fused with agonists or antagonists of cell-surface receptors, or cellular adhesion molecules to further modulate MPD intracellular effects. In another aspect of the invention, interleukins can be situated between the preferred MPD polypeptide fragment and other fusion polypeptide domains.

[0066] The MPD polypeptides of the invention can also include a localization sequence to direct the polypeptide to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins. A polynucleotide encoding a localization sequence, or signal sequence, can be ligated or fused at the 5′ terminus of a polynucleotide encoding an MPD polypeptide such that the signal peptide is located at the amino terminal end of the resulting fusion polynucleotide/polypeptide. In eukaryotes, the signal peptide functions to transport a polypeptide across the endoplasmic reticulum. The secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or the external environment. Signal peptides include pre-pro peptides that contain a proteolytic enzyme recognition site.

[0067] The localization sequence can be a nuclear-, an endoplasmic reticulum-, a peroxisome-, or a mitochondrial-localization sequence, or a localized protein. Localization sequences can be targeting sequences that are described, for example, in “Protein Targeting”, chapter 35 of Stryer, L., Biochemistry (4^(th) ed.). W. H. Freeman, 1995. Some important localization sequences include those targeting the nucleus (e.g., KKKRK (SEQ ID NO:38)), mitochondria (MLRTSSLFRRRVQPSLFRNILRLQST (SEQ ID NO:39)), endoplasmic reticulum (KDEL (SEQ ID NO:40)), peroxisome (SKF), plasma membrane (CAAX (SEQ ID NO:41), CC, CXC, or CCXX (SEQ ID NO:42)), cytoplasmic side of plasma membrane (fusion to SNAP-25), or the Golgi apparatus (fusion to furin).

[0068] In another embodiment, a polypeptide of the invention or fragments thereof may be fused to carrier molecules such as immunoglobulins for a variety of purposes including increasing the valency of polypeptide binding sites. As an example, fragments of the polypeptide may be fused through linker sequences to the Fc portion of an immunoglobulin. For a bivalent form of the polypeptide, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a polypeptide-IgM fusion would generate a decavalent form of the polypeptide of the invention. In one embodiment, the invention provides a fusion polypeptide having an Fc polypeptide domain and an MPD polypeptide sequence SEQ ID NO:6 beginning at an amino acid between and including residues 1 and 43 to 148; SEQ ID NO:8 beginning at an amino acid between and including residues 1 and 38 to 366; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO: 14 beginning at an amino acid between and including residues 1 and 84 to 622; SEQ ID NO: 14 beginning at an amino acid between and including residues 1 and 299 to 622; SEQ ID NO: 16; SEQ ID NO:21 from about residue 1 to 701; SEQ ID NO:23; SEQ ID NO:24 beginning at an amino acid between and including residues 1 and 278 to 435; SEQ ID NO:25 from about residue 1 to 627; and SEQ ID NO:26 beginning at an amino acid between and including residues 1 and 224 to 383.

[0069] The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising any or all of the CH domains of the Fc region. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Preferred polypeptides comprise an Fc polypeptide derived from a human IgGl antibody. As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion polypeptides comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction of Immunoglobulin Fusion Polypeptides”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992). Methods for preparation and use of immunoglobulin-based oligomers are known in the art. One embodiment of the invention is directed to a dimer comprising two fusion polypeptides created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fc fusion polypeptide is inserted into an appropriate expression vector. Polypeptide/Fc fusion polypeptides are expressed in host cells transformed or transfected with the recombinant expression vector or recombinant polynucleotide encoding the fusion polypeptide, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules. One suitable Fc polypeptide, described in PCr application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terninus of the Fc region of a human IgGl antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al., (EMBO J. 13:3992, 1994) incorporated herein by reference. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. The above-described fusion polypeptides comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Polypeptide A or Polypeptide G columns. In other embodiments, the polypeptides of the invention can be substituted for the variable portion of an antibody heavy or light chain. If fusion polypeptides are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four MPD polypeptides or fragments thereof.

[0070] Another method for preparing the oligomers of the invention involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization (dimers and trimers) of the polypeptides in which they are found. Leucine zippers were originally identified in several DNA-binding polypeptides (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different polypeptides. The zipper domain comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids.

[0071] A chimeric or fusion polypeptide of the invention can be produced by standard recombinant DNA techniques. In one embodiment, polynucleotide fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example, by employing blunt-ended or stagger-ended terinini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).

[0072] The invention further includes polypeptides with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems (e.g., COS-1 or CHO cells) can be similar to or significantly different from a native polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of polypeptides of the invention in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Further, a given preparation can include multiple differentially glycosylated species of the polypeptide. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase.

[0073] In another embodiment, modifications in the polypeptide or polynucleotide can be made using known techniques. Modifications of interest in the polypeptide sequences may include the alteration, substitution, replacement, insertion, or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule, an alteration which may involve preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation. Techniques for such alteration, substitution, replacement, insertion, or deletion are known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). As another example, N-glycosylation sites in a polypeptide's extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro, and Y is Ser or Thr. Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Alternatively, the Ser or Thr can by replaced with another amino acid, such as Ala. Known procedures for inactivating N-glycosylation sites in polypeptides include those described in U.S. Pat. No. 5,071,972 and EP 276,846, hereby incorporated by reference.

[0074] Additional variants within the scope of the invention include polypeptides that can be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives can be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein. Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the polypeptide.

[0075] The invention also provides polynucleotides encoding MPD polypeptides. The term “polynucleotide” refers to a polymeric form of nucleotides of at least 10 bases in length. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. The term includes single and double stranded forms of DNA or RNA. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The polynucleotides of the invention include full-length genes and cDNA molecules as well as a combination of fragments thereof. The polynucleotides of the invention are preferentially derived from human sources, but the invention includes those derived from nonhuman species, as well.

[0076] By “isolated polynucleotide” is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant polynucleotide molecule, which is incorporated into a vector, e.g., an expression vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.

[0077] An MPD polynucleotide of the invention (1) encodes a polypeptide comprising a sequence as set forth in SEQ ID NO: 1-26 or 27 or a fragment thereof; (2) has a sequence complementary to a (1); (3) polynucleotides that specifically hybridize to the polynucleotide of (1) under moderate to highly stringent conditions; and (4) polynucleotides of (1)-(3) wherein T can also be U (e.g., RNA sequences). Also encompassed by the invention are homologs of an MPD polynucleotide of the invention. These polynucleotides can be identified in several ways, including isolation of genomic or cDNA molecules from a suitable source, or computer searches of available sequence databases. Oligonucleotides or polynucleotides corresponding to the amino acid sequences described herein can be used as probes or primers for the isolation of polynucleotide homologs or as query sequences for database searches. Degenerate oligonucleotide sequences can be obtained by “back-translation” from the amino acid sequences of the invention. The polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding an MPD polypeptide. Oligonucleotides that define the desired termini of a target polynucleotide molecule are employed as 5′ and 3′ primers. Accordingly, fragments of the polynucleotides of the invention are useful as probes and primers to identify or amplify related sequence or obtain full-length sequences of an MPD of the invention. The oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are known in the art (see, e.g., PCR Protocols: A Guide to Methods ayzd Applicatioits, Innis et. al., eds., Academic Press, Inc. (1990)).

[0078] The invention also includes polynucleotides and oligonucleotides that hybridize under reduced stringency conditions, more preferably moderately stringent conditions, and most preferably highly stringent conditions, to MPD polynucleotides. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the polynucleotide. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6×SSC, and a hybridization temperature of about 55° C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42° C.), and washing conditions of about 60° C., in 0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68° C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al., 1989). When hybridizing a nucleic acid to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementary. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10° C. less than the melting temperature (T_(m)) of the hybrid, where T_(m) is determined according to the following equations. For hybrids less than 18 base pairs in length, T_(m) (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids above 18 base pairs in length, T_(m) (° C.)=81.5+16.6(log₁₀ [Na⁺])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na⁺] is the concentration of sodium ions in the hybridization buffer ([Na⁺] for 1×SSC=0.165M). Preferably, each such hybridizing nucleic acid has a length that is at least 25% (more preferably at least 50%, 60%, or 70%, and most preferably at least 80%) of the length of a polynucleotide of the invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, or at least 99%, and most preferably at least 99.5%) with a polynucleotide of the invention to which it hybridizes.

[0079] “Conservatively modified variants” applies to both polypeptide and polynucleotide. With respect to particular polynucleotide, conservatively modified variants refer to codons in the polynucleotide which encode identical or essentially identical amino acids. Because of the degeneracy of the genetic code, a large number of functionally identical polynucleotides encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such variations are “silent variations,” which are one species of conservatively modified variations. Every polynucleotide sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a polynucleotide (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0080] The invention also provides methodology for analysis of polynucleotides of the invention on “DNA chips” as described in Hacia et al., Nature Genetics, 14:441-447 (1996). For example, high-density arrays of oligonucleotides comprising a sequence encoding an MPD polypeptide, fragment, or a variant or mutant thereof are applied and immobilized to the chip and can be used to detect sequence variations in a population. Polynucleotides in a test sample are hybridized to the immobilized oligonucleotides. The hybridization profile of the target polynucleotide to the immobilized probe is quantitated and compared to a reference profile. The resulting genetic information can be used in molecular diagnosis. The density of oligonucleotides on DNA chips can be modified as needed.

[0081] The invention also provides genes corresponding to the polynucleotides disclosed herein. “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA molecules are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5′ and 3′ untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genoric libraries or other sources of genomic materials.

[0082] Expression, isolation, and purification of the polypeptides and fragments of the invention can be accomplished by any suitable technique, including but not limited to the following methods.

[0083] The isolated polynucleotides of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufinan et al., Nucleic Acids Res. 19:4485 (1991); and Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985, and Supplements), in order to produce a polypeptide of the invention recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant polypeptides are also known and are exemplified in R. Kaufman, Methods in Enzymology 185:537 (1990). As defined herein “operably linked” means that an isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the polypeptide encoded by the polynucleotide is expressed by a host cell which has been transformed (transfected) with the vector or polynucleotide operably linked to the control sequence.

[0084] In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. The choice of signal peptide or leader can depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced. Examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin (IL)-7 (see, U.S. Pat. No. 4,965,195); the signal sequence for IL-2 receptor (see, Cosman et al., Nature 312:768, 1984); the IL-4 receptor signal peptide (see, EP 367,566); the type I IL-1 receptor signal peptide (see, U.S. Pat. No. 4,968,607); and the type II IL-1 receptor signal peptide (see, EP 460,846). A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of a polypeptide from the cell. A polypeptide preparation can include a mixture of polypeptide molecules having different N-terminal amino acids, resulting from cleavage, of the signal peptide at more than one site.

[0085] Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine or Lipofectamine-Plus lipid reagent (Gibco/BRL), can be used to transfect cells (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology 185:487, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub et al., Proc. Natl. Acad Sci. USA 77:4216, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector are selected on the basis of resistance to these compounds.

[0086] Alternatively, gene products can be obtained via homologous recombination, or “gene targeting” techniques. Such techniques employ the introduction of exogenous transcription control elements (such as the CMV promoter or the like) in a particular predetermined site on the genome, to induce expression of an endogenous gene encoding an MPD polypeptide of the invention. The location of integration into a host chromosome or genome can be easily determined by one of skill in the art, given the known location and sequence of the gene. In a preferred embodiment, the invention also contemplates the introduction of exogenous transcriptional control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product. The practice of homologous recombination or gene targeting is explained by Schirnke, et al. “Amplification of Genes in Somatic Mammalian cells,” Methods in Enzymology 151:85 (1987), and by Capecchi, et al., “The New Mouse Genetics: Altering the Genoyzie by Gene Targeting,” TG 5:70 (1989).

[0087] Suitable host cells for expression of the polypeptide include eukaryotic and procaryotic cells. Mammalian host cells include, for example, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see, McMahan et al. EMBO J. 10:2821, 1991), human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomizyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides. Potentially suitable bacterial strains include, for example, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional polypeptide. Such covalent attachments may be accomplished using known chemical or enzymatic methods. The polypeptide may also be produced by operably linking a polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), as well as methods described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Technology 6:47 (1988), incorporated herein by reference. Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from nucleic acid constructs disclosed herein. A host cell that comprises an isolated polynucleotide of the invention, preferably operably linked to at least one expression control sequence, is a “recombinant host cell”.

[0088] Any method, which neutralizes MPD polypeptides or inhibits expression (either transcription or translation) of an MPD polynucleotide can be used to reduce the biological activities of MPD polypeptides.

[0089] In one embodiment, antagonists can be designed to reduce the level of endogenous MPD expression, e.g., using known antisense or ribozyme approaches to inhibit or prevent translation of MPD mRNA transcripts; triple helix approaches to inhibit transcription of MPD genes; or targeted homologous recombination to inactivate or “knock out” the MPD genes or their endogenous promoters or enhancer elements. Such antisense, ribozyme, and triple helix antagonists may be designed to reduce or inhibit either unimpaired or, if appropriate, mutant MPD activity.

[0090] Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing polypeptide translation. Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to a mRNA having an MPD polynucleotide sequence. Absolute complementary, although preferred, is not required. Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to, and including, the AUG initiation codon, should work most efficiently at inhibiting translation. Antisense nucleic acids are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. The oligonucleotides can be DNA, RNA, chimeric mixtures, derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, and the like. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. 84:648, 1987; PCT Publication No. WO88/09810), or hybridization-triggered cleavage agents or intercalating agents (see, e.g., Zon, Pharm. Res. 5:539, 1988). The antisense molecules are delivered to cells, which express a transcript having an MPD polynucleotide sequence in vivo by, for example, direct injection into the tissue or cell derivation site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically. Preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.

[0091] Ribozyme molecules designed to catalytically cleave mRNA transcripts having an MPD polynucleotide sequence prevent translation of MPD mRNA (see, e.g., PCT International Publication WO90/11364; U.S. Pat. No. 5,824,519). Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA. Because ribozymes are sequence-specific, only mRNAs with particular sequences are inactivated. There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type. Tetrahymena-type ribozymes recognize sequences, which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymenatype ribozymes. As in the antisense approach, ribozymes can be composed of modified oligonucleotides and delivered using using a DNA construct “encoding” the ribozyme under the control of a strong constitutive pol III or pol II promoter.

[0092] Alternatively, endogenous MPD expression can be reduced by targeting DNA sequences complementary to a regulatory region of the target gene (e.g., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene (see generally, Helene, Anticancer Drug Des., 6(6), 569, 1991; Helene, et al., Ann. N.Y. Acad. Sci., 660:27, 1992; and Maher, Bioassays 14(12), 807, 1992).

[0093] Antisense, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules and include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides such as, for example, solid phase phosphoramidite chemical synthesis using an automated DNA synthesizer available from Biosearch, Applied Biosystems. Phosphorothioate oligonucleotides may be synthesized by the method of Stein et al., Nucl. Acids Res. 16:3209, 1988. Methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448, 1988). Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.

[0094] Endogenous gene expression can also be reduced by inactivating or “knocking out” the target gene or its promoter using targeted homologous recombination (see, e.g., Smithies, et al., Nature 317:230, 1985; Thomas and Capecchi, Cell 51, 503, 1987; Thompson, et al., Cell 5, 313, 1989; each of which is incorporated by reference herein in its entirety). For example, a mutant non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene can be used, with or without a selectable marker and/or a negative selectable marker. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited where modifications to embryonic stem cells can be used to generate non-human animal offspring with an inactive target gene (e.g., see Thomas and Capecchi, 1987 and Thompson, 1989, supra; see also the “RNA interference” (“RNAi”) technique of Grishok et al., Science 287 (5462): 2494, 2000), and Dernburg et al., Genes Dev. 14 (13): 1578, 2000).

[0095] As used herein, a “transgenic animal” is an animal that includes a transgene that is inserted into an embryonal cell and becomes a part of the genome of the animal that develops from that cell, or an offspring of such an animal. Any non-human animal that can be produced by transgenic technology is included in the invention, although mammals are preferred. Preferred mammals include non-human primates, sheep, goats, horses, cattle, pigs, rabbits, and rodents, such as, guinea pigs, hamsters, rats, gerbils, and mice.

[0096] A “transgene” is a polynucleotide that comprises one or more selected sequences (e.g., encoding ribozymes that cleave MPD mRNA, encoding an antisense molecule to an MPD mRNA, encoding a mutant MPD sequence, and the like) to be expressed in a transgenic animal. The polynucleotide is partly or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but which is designed to be inserted into the animal's genome at a location which differs from that of the natural gene. A transgene may include one or more promoters and any other DNA sequences, such as introns, necessary for expression of the selected DNA, all operably linked to the selected DNA, and may include an enhancer sequence.

[0097] The transgenic animal can be used in order to identify the impact of increased or decreased MPD levels on a particular pathway or phenotype. Protocols useful in producing such transgenic animals are known in the art (see, e.g., Brinster, et al., Proc. Natl. Acad Sci. USA 82:4438, 1985; Jaenisch, Proc. Natl. Acad. Sci. USA 73:1260, 1976; Hogan, et al., 1986, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Jahner, et al., Proc. Natl. Acad. Sci. USA 82:6927, 1985; Van der Putten, et al., Proc Natl. Acad. Sci. USA 82:6148; Steward, et al., EMBO J., 6:383, 1987; Jahner, et al., Nature, 298:623, 1982).

[0098] In another embodiment, Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. The MPD polypeptides, fragments, variants, fusion polypeptides, and the like, as set forth above, can be employed as “immunogens” in producing antibodies immunoreactive therewith. Such antibodies specifically bind to the polypeptides via the antigenbinding sites of the antibody. Specifically binding antibodies are those that will specifically recognize and bind with MPD polypeptides, homologues, and variants, but not with other molecules. In a preferred embodiment, the antibodies are specific for polypeptides having an MPD amino acid sequence of the invention and do not cross-react with other polypeptides.

[0099] More specifically, the polypeptides, fragment, variants, fusion polypeptides, and the like contain antigenic determinants or epitopes that elicit the formation of antibodies. These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon polypeptide folding. Epitopes can be identified by any of the methods known in the art. Additionally, epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.

[0100] Both polyclonal and monoclonal antibodies to the polypeptides of the invention can be prepared by conventional techniques. See, for example, Monocloital Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler and Milstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026, 1983); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. For the production of antibodies, various host animals may be immunized by injection with an MPD polypeptide, fragment, variant, or mutants thereof. Such host animals may include, but are not limited to, rabbits, mice, and rats, to name a few. Various adjuants may be used to increase the immunological response. Depending on the host species, such adjutants include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. The monoclonal antibodies can be recovered by conventional techniques. Such monoclonal antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

[0101] In addition, techniques developed for the production of “chimeric antibodies” (Takeda et al., Nature, 314:452, 1985) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a porcine mAb and a human immunoglobulin constant region. The monoclonal antibodies of the invention also include humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, Can, 1993). Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incorporated by reference herein. Preferably, for use in humans, the antibodies are human or humanized; techniques for creating such human antibodies are also known. Transgenic animals for making human antibodies are available from, for example, Medarex Inc. (Princeton, N.J.) and Abgenix Inc. (Fremont, Calif.).

[0102] Antibody fragments, which recognize specific epitopes, may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the (ab′)₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., Science, 246:1275, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879, 1988; and Ward et al., Nature 334:544, 1989) can also be adapted to produce single chain antibodies against polypeptides containing MPD amino acid sequences. In addition, antibodies to the MPD polypeptide can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” an MPD polypeptide and that may bind to the MPD polypeptide using techniques known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J 7(5):437, 1993; and Nissinoff, J. Immunol. 147(8):2429, 1991).

[0103] Screening procedures to identify such antibodies are known, and can involve immunoaffinity chromatography, for example. Antibodies can be screened for agonistic (i.e., ligand-mimicking) properties. Such antibodies, upon binding to an MPD polypeptide on the cell surface, can induce biological effects (e.g., transduction of biological signals) similar to the biological effects induced when the naturally occurring MPD binding partner binds to the polypeptide on the cell surface. Agonistic antibodies can be used to induce MPD mediated capitulatory pathways or intercellular communication.

[0104] In addition, antibodies that block binding of a polypeptide having an MPD polypeptide sequence of the invention to its binding partner or substrate can be used to inhibit MPD polypeptide mediated intercellular communication or co-stimulation that results from such binding and/or to identify integrin cognates of an MPD polypeptide. Such blocking antibodies can be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of an MPD polypeptide to certain cells expressing a binding partner (e.g., an integrin) to the polypeptide. Alternatively, blocking antibodies can be identified in assays for the ability to inhibit a biological effect that results from binding of an MPD polypeptide to target cells. In one embodiment, a flow cytometric integrin mAb based binding inhibition assay is used to show binding of MPDdis-Fc polypeptides to integrins expressed on the surface of endothelial cells. Human endothelial cells can be used in such assay. Human endothelial cells express α_(v)β₃, α_(v)β₅, β₁, β₄, α₁, α₂, α₃, α₄, α₅, and α₆ integrins. An MPDdis-Fc polypeptide is contacted with the endothelial cells. Monoclonal antibodies specific for human integrins α_(v)β₃ (LM609, anti-CD51/61, Chemicon, Temecula, Calif.; Brooks et al, Science 264:569, 1994), α₂β₁ (BHA2.1, anti-CD49b, Chemicon; Wang et al., Mol. Biol. of the Cell 9:865, 1998), α₅β₁ (SAM-1, anti-CD49e, Biodesign; A. te Velde et al., J. Immunol. 140:1548, 1988), α₃β₁ (ASC-6, anti-CD49c, Chemicon; Pattaramalai et al., Exp. Cell. Res. 222: 281, 1996), α₄β₁ (HP2/1, anti-CD49d, Immunotech, Marseilles, France; Workshop of the 4^(th) International Conference on Human Leukocyte Differentiation Antigens, Vienna Austria, 1989, workshop number p091), α₆β₁ (GoH3, anti-CD49f, Immunotech; Workshop 4^(th) International Conference on Human Leukocyte Differentiation Antigens, workshop number p055), α₆β₄ (439-9B, anti-CD104, Pharmingen, San Diego, Calif.; Schlossman et al., 1995 Leukocyte Typing V: White Cell Differentiation Antigens. Oxford University Press, New York), and α_(v)β₅. (MAB 1961, Chemicon; Weinaker, et al., J. Biol. Chem. 269:6940, 1994) were shown to bind specifically to HMVEC-d. Each of these antibodies is known to specifically block binding of the indicated integrin to its ligands (e.g., fibronectin, vitronectin, fibrinogen). The ability of integrin mAbs to inhibit the binding of MPDdis-Fc polypeptides reveals which integrins the disintegrin domain of an MPD polypeptide binds and, indirectly, which integrin binding activities the disintegrin domains are able to antagonize. MPDdis-Fc polypeptides that bind to select integrins are further tested for the ability to disrupt integrin-ligand interactions and to modulate endothelial cell function, angiogenesis, and other biological activities in vitro and in vivo.

[0105] Disorders caused or exacerbated (directly or indirectly) by the interaction of MPD polypeptides with a cell surface-binding partner can thus be treated. A therapeutic method involves in vivo administration of a blocking antibody to a subject in an amount effective to inhibit MPD binding-mediated biological activity. As used herein, a “subject” can be any animal, preferably a mammal (e.g., canine, feline, bovine, porcine, equine, primates, and the like), and most preferably a human. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In one embodiment, an antigen-binding antibody fragment is employed. Compositions comprising an antibody against an MPD polypeptide, and a physiologically acceptable diluent, excipient, or carrier, are provided herein.

[0106] Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent attached to an anti-MPD polypeptide antibody. The conjugates find use in in vitro or in vivo procedures. The antibodies of the invention can also be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or ill vivo. The antibodies also can be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.

[0107] In another embodiment, rational drug design is used to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, e.g., substrates, binding agents, inhibitors, agonists, antagonists, and the like. The methods provided herein can be used to fashion or identify agents which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (Hodgson J, Biotechnology 9:19, 1991, incorporated herein by reference). In one approach, the three-dimensional structure of a polypeptide of the invention, a ligand or binding partner, or of a polypeptide-binding partner complex, is determined by x-ray crystallography, by nuclear magnetic resonance, or by computer homology modeling or, most typically, by a combination of these approaches. Relevant structural information is used to design analogous molecules, to identify efficient inhibitors, or to identify small molecules that may bind to a polypeptide of the invention. The use of ADAM polypeptide structural information, preferably MPD structural information, in molecular modeling software systems provides for the design of inhibitors or binding agents useful in modulating MPD activity. A particular method of the invention comprises analyzing the three dimensional structure of MPD polypeptides for likely binding sites of substrates or ligands, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described further herein. Examples of algorithms, software, and methods for modeling substrates or binding agents based upon the three-dimensional structure of a protein are described in PCT publication WO107579A2, the disclosure of which is incorporated herein.

[0108] It is also possible to isolate a target-specific antibody, selected by a functional assay, as described further herein, and then to solve its crystal structure thus yielding a pharmacore upon which subsequent drug design can be based. It is possible to bypass polypeptide crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.

[0109] The invention provides methods for identifying agents that modulate MPD polypeptide activity or expression. Such methods included contacting a sample containing an MPD polypeptide or polynucleotide with a test agent under conditions that allow for the test agent and the polypeptide or polynucleotide to interact and measuring the expression or activity of an MPD polypeptide in the presence or absence of the test agent.

[0110] In one embodiment, a cell containing an MPD polynucleotide is contacted with a test agent under conditions such that the cell and test agent are allowed to interact. Such conditions typically include normal cell culture conditions consistent with the particular cell type being utilized and which are known in the art. It may be desirable to allow the test agent and cell to interact under conditions associated with increased temperature or in the presence of regents that facilitate the uptake of the test agent by the cell. A control is treated similarly but in the absence of the test agent. Alternatively, the MPD activity or expression may be measured prior to contact with the test agent (e.g., the standard or control measurement) and then again following contact with the test agent. The treated cell is then compared to the control and a difference in the expression or activity of MPD compared to the control is indicative of an agent that modulates MPD activity or expression.

[0111] When MPD expression is being measured, detecting the amount of mRNA encoding an MPD polypeptide in the cell can be quantified by, for example, PCR or Northern blot. Where a change in the amount of MPD polypeptide in the sample is being measured, detecting or quantifying MPD polypeptide can be performed using anti-MPD antibodies using known techniques.

[0112] A test agent can be any molecule typically used in the modulation of protein activity or expression and includes, for example, small molecules, chemicals, peptidomimetics, antibodies, peptides, polynucleotides (e.g., antisense or ribozyme molecules), and the like. Accordingly, agents developed by computer based drug design can be tested in the laboratory using the assay and methods described herein to determine the activity of the agent on the modulation of MPD activity or expression. Modulation of MPD includes an increase or decrease in activity or expression.

[0113] An MPD polypeptide of the invention (including fragments, variants, oligomers, and other forms) are useful in a variety of assays. For example, an MPD polypeptide of the invention can be used to identify binding partners of members of the ADAM family of polypeptides, which can also be used to modulate intercellular communication, co-stimulation, or immune cell activity. Alternatively, they can be used to identify non-binding-partner molecules or substances that modulate intercellular communication, co-stimulatory pathways, or immune cell activity.

[0114] MPD polypeptides and fragments thereof can be used to identify binding partners. For example, they can be tested for the ability to bind a candidate-binding partner in any suitable assay, such as a conventional binding assay. To illustrate, an MPD polypeptide or fragment thereof can be labeled with a detectable molecule (e.g., a radionuclide, a chromophore, and an enzyme that catalyzes a colorimetric or fluorometric reaction and the like). The labeled polypeptide is contacted with cells expressing the candidate-binding partner. The cells then are washed to remove unbound-labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.

[0115] In one embodiment, a binding partner integrin is identified by the use of anti-integrin antibodies. The ability of integrin mAbs to inhibit the binding of MPDdis-Fc polypeptides reveals which integrin the disintegrin domain binds and, indirectly, which integrin binding activities the disintegrin domain is able to antagonize. MPDdis-Fc polypeptides that bind to select integrins are further tested for the ability to disrupt integrin-ligand interactions and to modulate endothelial cell function, angiogenesis, and other biological activities ill vitro and in vivo.

[0116] In another example of a binding assay a recombinant expression vector containing the candidate binding partner cDNA is transfected into CV1-EBNA-1 cells. The cells are incubated for 1 hour at 37° C. with various concentrations of, for example, a soluble MPD polypeptide/Fc fusion polypeptide. Cells are washed and incubated with a constant saturating concentration of a ¹²⁵I-mouse anti-human IgG. After washing, cells are released via trypsinization. The mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, Pa. The antibody will bind to the Fc portion of any Fc polypeptide that has bound to the cells. Cell-bound ¹25I-antibody is quantified on a Packard Autogamma counter.

[0117] Where an MPD polypeptide binds or potentially binds to another polypeptide (e.g., in a receptor-ligand interaction), the MPD polynucleotide can also be used in interaction trap assays (see, e.g., Gyuris et al., Cell 75:791, 1993) to identify polynucleotides encoding the other polypeptide with which binding occurs or to identify inhibitors of the binding interaction. Polypeptides involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

[0118] Another type of suitable binding assay is a competitive binding assay. To illustrate, biological activity of a variant can be determined by assaying for the variant's ability to compete with the native polypeptide for binding to the candidate-binding partner. Competitive binding assays can be performed by conventional methodology. Reagents that can be employed in competitive binding assays include a radiolabeled MPD fragment or variant and intact cells expressing MPD (endogenous or recombinant) on the cell surface. Instead of intact cells, one could substitute a soluble binding partner/Fc fusion polypeptide bound to a solid phase through the interaction of Polypeptide A or Polypeptide G (on the solid phase) with the Fc moiety. Chromatography columns that contain Polypeptide A and G include those available from Pharmacia Biotech, Inc., Piscataway, N.J.

[0119] Enzymatic assays can be used to measure metalloproteinase activity of MPD polypeptides. For example, the activity of an MPD metalloproteinase polypeptide can be measure by incubating a fluorescently labeled MPD metalloproteinase substrate with an MPD metalloproteinase polypeptide and measuring a change in the fluorescence or location of fluorescence. Development of fluorescently labeled substrates in known in the art.

[0120] The influence of MPD polypeptides, MPD fragments and antibodies on intercellular communication, co-stimulation, integrin binding, endothelial cell migration, angiogenesis or immune cell activity can be assayed by contacting a cell or a group of cells with a polynucleotide, polypeptide, agonist or antagonist, to induce, enhance, suppress, or arrest cellular communication, costimulation, integrin binding, endothelial cell migration, angiogenesis or activity in the target cells. Identification of MPD polypeptides, agonists or antagonists can be carried out via a variety of assays known to those skilled in the art. Included in such assays are those that evaluate the ability of an MPD polypeptide to influence intercellular communication, co-stimulation, integrin binding, endothelial cell migration, or angiogenesis. Such an assay would involve, for example, the analysis of cell-cell interactions (e.g., through integrin-related binding) in the presence of an MPD polypeptide or soluble disintegrin fragment thereof. In such an assay, one would determine a rate of cell-cell interaction, cell matrix interaction, or integrin associated binding in the presence of a polypeptide having an MPD sequence and then determine if such binding or interaction is altered in the presence of, e.g., a soluble disintegrin MPD (MPDdis) sequence. Exemplary assays for this aspect of the invention includes endothelial migration assays. Other assays are known in the art.

[0121] In another aspect, the invention provides a method of detecting the ability of a test agent to affect the cell-ell interaction, cell-matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity of the test agent on a cell or culture. In this aspect, the method comprises: (1) contacting a first group of target cells with a test agent including a polypeptide comprising an MPD sequence (e.g., SEQ ID NO: 1-27; or a soluble MPD disintegrin polypeptide), a ligand or receptor for an MPD polypeptide, or fragment thereof, under conditions appropriate to the particular assay being used; (2) measuring the net rate of cell-cell interaction, cell-matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity among the target cells; and (3) observing the net rate of cell-cell interaction, cell-matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity among control cells containing an MPD polypeptide ligand or fragments thereof, in the absence of a test agent, under otherwise identical conditions as the first group of cells. In this embodiment, the net rate of intercellular communication or co-stimulation in the control cells is compared to that of the cells treated with both an MPD molecule as well as a test agent. The comparison will provide a difference in the net rate of cell-cell interaction, cell matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity indicative of an agent that modulates MPD activity. The test agent can function as an effector by either activating or up-regulating, or by inhibiting or down-regulating cell-cell interaction, cell-matrix interaction, integrin associated binding, endothelial cell migratory activity, or angiogenic activity.

[0122] A polypeptide of the invention may exhibit cytokine production or inhibition activity, cell proliferation (either inducing or inhibiting), or cell differentiation (either inducing or inhibiting) activity. Many polypeptide factors discovered to date, including all known cytokines, have exhibited activity in one or more cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a polypeptide of the invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of an MPD polypeptide of the invention may be measured by the following methods:

[0123] Assays for T-cell or thymocyte proliferation include, without limitation, those described in: Current Protocols in immunology, Ed. by Coligan et al., Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494, 1986; Bertagnolli et al., J. Immunol. 145:1706, 1990; Bertagnolli et al., Cell. Immunol. 133:327, 1991; Bertagnolli, et al., J. Immunol. 149:3778, 1992; Bowman et al., J. Immunol. 152:1756, 1994.

[0124] Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, Vol 1 pp. 3.12.1-3.12.14, and Measurement of mouse and human Interferon y, Schreiber, R. D. Vol 1 pp. 6.8.1-6.8.8. In Current Protocols in Immunology. E. M. Coligan eds. John Wiley and Sons, Toronto. 1994; Coligan eds., John Wiley and Sons, Toronto, 1994.

[0125] Assays for proliferation and differentiation of hematopoietic and lymphonoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly et al., In Current Protocols in Immunology. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205, 1991; Moreau et al., Nature 336:690, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931, 1983; Measurement of mouse and human interleukin 6, Nordan, R. In Current Protocols in Immunology. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857, 1986; Measurement of human Interleukin 11, Bennett et al., In Current Protocols in Immunology. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9, Ciarletta et al., In Current Protocols in Immunology. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

[0126] Assays for T-cell clone responses to antigens (which will identify, among others, polypeptides that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Inuunology, Coligan eds., Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, in vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091, 1980; Weinberger et al., Eur. J. Immun. 11:405, 1981; Takai et al., J. Immunol. 137:3494, 1986; Takai et al., J. Immunol. 140:508, 1988.

[0127] Assays for thymocyte or splenocyte cytotoxicity include, without limitation, Current Protocols in Immunology, Coligan eds., Pub. Greene Publishing Associates and Wiley-Interscience (hi vitro assays for Mouse Lymphocyte Function pp. 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488, 1981; Hefrmann et al., J.Immunol. 128:1968, 1982; Handa et al., J.Immunol. 135:1564, 1985; Takai et al., J.Immunol. 137:3494, 1986; Takai et al., J.Immunol. 140:508, 1988; Bowman et al., J.Virol. 61:1992; Bertagnolli et al., Cell. mm. 133:327, 1991; Brown et al., J.Immun. 153:3079, 1994.

[0128] Assays for T-cell-dependent IgG responses and isotype switching (which will identify, among others, polypeptides that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, Wiley and Sons, Toronto. 1994.

[0129] Mixed lymphocyte reaction (MLR) assays (which will identify, among others, polypeptides that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Coligan eds., Pub. Greene Publishing Associates and Wiley-Interscience (In vitro assays for Mouse Lymphocyte Function pp 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., 1986, supra; Takai et al., 1988, supra; Bertagnolli et al., J. Immunol. 149:3778, 1992.

[0130] Dendritic cell-dependent assays (which will identify, among others, polypeptides expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536, 1995; Inaba et al., J. of Exp. Med. 173:549, 1991; Macatonia et al., J. Immunol. 154:5071, 1995; Porgador et al., J. of Exp. Med. 182:255, 1995; Nair et al., J. Virol. 67:4062, 1993; Huang et al., Science 264:961, 1994; Macatonia et al., J. of Exp. Med. 169:1255, 1989; Bhardwaj et al., S. Clin. Invest. 94:797, 1994; and Inaba et al., J. of Exp. Med. 172:631, 1990.

[0131] Assays for lymphocyte survival apoptosis (which will identify, among others, polypeptides that prevent apoptosis after superantigen induction and polypeptides that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795, 1992; Gorczyca et al., Leukemia 7:659, 1993; Gorczyca et al, Cancer Research 53:1945, 1993; Itoh et al., Cell 66:233, 1991; Zacharchuk, J. Immunol. 145:4037, 1990; Zamai et al., Cytometry 14:891, 1993; Gorczyca et al., Int. J. of Oncology 1:639, 1992.

[0132] Assays for polypeptides that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111, 1994; Fine et al., Cell. Immunol. 155:111, 1994; Galy et al., Blood 85:2770, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548, 1991.

[0133] Assays for embronic stem cell differentiation (which will identify, among others, polypeptides that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cell. Biol. 15:141, 1995; Keller et al., Mol. and Cell. Biol. 13:473, 1993; McClanahan et al., Blood 81:2903, 1993.

[0134] Assays for stem cell survival and differentiation (which will identify, among others, polypeptides that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. L. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Exp. Hematol. 22:353, 1994; Cobblestone area forming cell assay, Ploemacher, In Culture of Hematopoietic Cells. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer et al. In Culture of Hematopoietic Cells. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, In Culture of Hematopoietic Cells. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

[0135] Assays for tissue generation activity include, without limitation, those described in: Patent Publication No. WO95/16035 (bone, cartilage, tendon); Patent Publication No. WO95/05846 (nerve, neuronal); Patent Publication No. WO91/07491 (skin, endothelium). Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, and Rovee, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

[0136] Assays for activity/inhibin activity include, without limitation, those described in: Vale et al., Endocrinol. 91:562, 1972; Ling et al., Nature 321:779, 1986; Vale et al., Nature 321:776, 1986; Mason et al., Nature 318:659, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091, 1986.

[0137] Assays for cell movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Coligan eds., Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of a and P Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140, 1995; Muller et al. Eur. J. Immunol. 25:1744; Gruber et al. J. Immunol. 152:5860, 1994; Johnston et al. J. Immunol. 153: 1762, 1994.

[0138] Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131, 1986; Burdick et al., Thrombosis Res. 45:413,1987; Humphrey et al., Fibrinolysis 5:71, 1991; Schaub, Prostaglandins 35:467, 1988.

[0139] Assays for receptor-ligand activity include, without limitation, those described in: Current Protocols in Immunology, Coligan eds., Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864, 1987; Bierer et al., J. Exp. Med. 168:1145, 1988; Rosenstein et al., J. Exp. Med. 169:149, 1989; Stoltenborg et al., J. Immunol. Methods 175:59, 1994; Stitt et al., Cell 80:661, 1995.

[0140] Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol. Chem. 270(32):18809, 1995; Miyaki et al. Oncogene 11:2547, 1995; Ozawa et al. Cell 63:1033, 1990.

[0141] A polynucleotide encoding a polypeptide having an MPD sequence provided by the invention can be used for numerous diagnostic or other useful purposes. A polynucleotide of the invention (e.g., a polynucleotide encoding SEQ ID NO:1-26 or 27) can be used as markers for tissues in which the corresponding polypeptide is preferentially expressed, as molecular weight markers on Southern gels, as chromosome markers or tags to identify chromosomes or to map related gene positions, to compare with endogenous DNA sequences in subjects to identify potential genetic disorders, as probes to hybridize and thus discover novel related polynucleotides, as a source of information to derive PCR primers for genetic fingerprinting, as a probe to “subtract-out” known polynucleotides in the process of discovering other novel nucleic acids, as an antigen to raise anti-DNA antibodies or elicit another immune response, and for gene therapy.

[0142] Probes and Primers. Among the uses of the disclosed MPD polynucleotides, and combinations of fragments thereof, is the use of fragments as probes or primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60 contiguous nucleotides of a DNA sequence. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al., 1989 and are described in detail above. Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for non-human genetic libraries. Such libraries would include but are not limited to cDNA libraries, genomnic libraries, and even electronic EST (express sequence tag) or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify nonhuman homologues of the MPD sequence identified herein.

[0143] Chromosome Mapping. The polynucleotides encoding MPD polypeptides, and the disclosed fragments and combinations of these polynucleotides, can be used by those skilled in the art using known techniques to identify the human chromosome to which these sequences map. Useful techniques include, but are not limited to, using the sequence or portions, including oligonucleotides, as a probe in various known techniques such as radiation hybrid mapping (high resolution), in situ hybridization to chromosome spreads (moderate resolution), and Southern blot hybridization to hybrid cell lines containing individual human chromosomes (low resolution). The following web site provides additional information about radiation hybrid mapping: www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/07-97.INTRO.html.

[0144] A polynucleotide encoding a polypeptide having an MPD polypeptide sequence of the invention, and the disclosed fragments and combinations of these polynucleotides can be used to analyze abnormalities associated with the genes corresponding to MPD polypeptides. This enables one to distinguish conditions in which this marker is rearranged or deleted. In addition, polynucleotides of the invention or a fragment thereof can be used as a positional marker to map other genes of unknown location. The polynucleotide can be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, genes (e.g., an MPD-associated disorder) corresponding to the polynucleotides of the invention. The polynucleotides and associated sequences disclosed herein permit the detection of defective genes, and the replacement thereof with normal genes. Defective genes can be detected in in vitro diagnostic assays, and by comparison of the polynucleotide sequences disclosed herein with that of a gene derived from a subject suspected of harboring a defect in this gene or having an MPD-associated disorder.

[0145] Uses of MPD polypeptides and peptide fragments thereof include, but are not limited to, the following: delivery agents; therapeutic and research reagents; molecular weight and isoelectric focusing markers; controls for peptide fragmentation; identification of unknown polypeptides; and preparation of antibodies.

[0146] The MPD polypeptides (e.g., SEQ ID NO:1-26 or 27) of the invention can be used as polypeptide purification reagents. For example, MPD polypeptides can be attached to a solid support material and used to purify its binding partners (e.g., an integrin molecule) by affinity chromatography. In particular embodiments, a polypeptide is attached to a solid support by conventional procedures. As one example, chromatography columns containing functional groups that will react with amino acid side chains of polypeptides are available (Pharmacia Biotech, Inc., Piscataway, N.J.). In an alternative, an MPD-Fc polypeptide is attached to Polypeptide A- or Polypeptide G-containing chromatography columns through interaction with the Fc moiety. The polypeptide also finds use in purifying or identifying cells that express a binding partner on the cell surface. Polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing the binding partner expressing cells are contacted with the solid phase having the polypeptides thereon. Cells expressing the binding partner on the cell surface bind to the polypeptides on the solid phase, and unbound cells then are washed away. Alternatively, the polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for binding partner expression. After incubation, unbound-labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined.

[0147] Carriers and Delivery Agents. The polypeptides also find use as carriers for delivering agents attached thereto to cells bearing identified binding partners (e.g., an integrin). The polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells in in vitro or in vivo procedures. Detectable (diagnostic) and therapeutic agents that can be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radio-nuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application. Among the toxins are ricin, abrin, diphtheria toxin, Pseudoinonas aenigiyosa exotoxin A, ribosomal inactivating polypeptides, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples of radionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu. Such agents can be attached to the polypeptide by any suitable conventional procedure. The polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the polypeptide or agent can be derivatized to generate or attach a desired reactive functional group. The derivatization can involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to polypeptides (Pierce Chemical Company, Rockford, Illinois). Of particular interest are soluble MPD disintegrins that can be used to target cells expressing a binding partner for the MPD disintegrin moiety (e.g., an integrin). Such soluble MPD disintegrins can be used to target reagents to cells expressing, for example, the disintegrin's cognate integrin. Similarly, and as discussed more fully below, antibodies specific for an MPD polypeptide can be labeled with a diagnostic or therapeutic agent and used to target the diagnostic or therapeutic to cells expressing an MPD polypeptide.

[0148] MPD polypeptides and MPD fragments (e.g., fragments having disintegrin and/or metalloproteinase activity) can be employed in modulating a biological activity of an ADAM polypeptide, particularly MPD polypeptide, in in vitro or in vivo procedures. Encompassed within the invention are domains of MPD polypeptides that act as modulators of native ADAM polypeptide function, including native MPD activity, when expressed as fragments or as components of fusion polypeptides. For example, a substantially purified polypeptide domain of the invention can be used to inhibit binding of an MPD polypeptide to endogenous binding partners. Such use effectively would block MPD interactions and inhibit MPD activities. In still another aspect of the invention, a soluble form of an MPD binding partner (e.g., a soluble integrin domain) is used to bind to, and competitively inhibit activation of the endogenous MPD polypeptide.

[0149] In another embodiment, the invention is directed to methods of inhibiting the binding of an integrin to its ligand, and thereby inhibiting the biological activity of the integrin, comprising contacting the integrin with an effective amount of an MPDdis polypeptide. The invention is further directed to methods of inhibiting endothelial cell migration and methods of inhibiting angiogenesis comprising administering an effective amount of an MPDdis polypeptide. In some embodiments the MPDdis polypeptide is in the form of a multimer, preferably a leucine zipper multimer or Fc polypeptide. Alternatively, substantially purified or modified MPD polypeptides of the invention can be administered to modulate interactions between MPD polypeptides and MPD binding partners that are not membrane-bound.

[0150] Antibodies that bind to MPD polypeptides can inhibit MPD polypeptide activity and may act as antagonists. For example, antibodies that specifically bind to one or more epitopes of an MPD polypeptide, or epitope of conserved variants of MPD polypeptides, or fragments can be used to inhibit MPD activity. By “specifically bind” means that an antibody to an MPD polypeptide or fragment thereof will not cross-react with unrelated polypeptides. Preferably such an antibody will not cross-react with other members of the ADAM family.

[0151] In an alternative aspect, the invention further encompasses the use of agonists of MPD activity to treat or ameliorate the symptoms of a disease for which increased disintegrin activity is beneficial. In a preferred aspect, the invention entails administering compositions comprising an MPD polynucleotide or fragment thereof or a polypeptide comprising an MPD amino acid sequence (e.g., SEQ ID NO:1-26 or 27) or fragment thereof. The administering may be to cells ill vitro, to cells ex vivo, to cells in vivo, and/or to a multicellular organism. Preferred therapeutic forms include soluble forms of an MPD having disintegrin activity. Such a soluble MPD disintegrin polypeptide will bind to its binding partner (e.g., an integrin) and stimulate a biological activity associated with the binding partner.

[0152] In still another aspect of the invention, the compositions comprise administering a polynucleotide encoding an MPD polypeptide for expression in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human subject for treatment of a dysfunction associated with aberrant (e.g., decreased) endogenous activity of an MPD polypeptide. Furthermore, the invention encompasses the administration of compounds found to increase the endogenous activity of polypeptides comprising an MPD amino acid sequence to cells and/or organisms. One example of compounds that increase MPD polypeptide activity are antibodies that bind to MPD polypeptides, preferably monoclonal antibodies, and increase or stimulate MPD polypeptide activity by causing constitutive intracellular signaling (or “ligand mimicking”), or by preventing the binding of a native inhibitor of MPD polypeptide activity.

[0153] Due to the multiplicity and interconnectedness of biological pathways and interactions, an MPD polypeptide, fragment, variant, antagonist, agonist, antibody, and binding partner of the invention can be useful for treating medical conditions and diseases associated with cell-cell and cell matrix interactions (e.g., integrin-mediated disorders), endothelial migration, angiogenesis, inflammation, cancer, allergy, reproductive, neurological and vascular conditions as described further herein. The therapeutic molecule or molecules to be used will depend on the etiology of the condition to be treated and the biological pathways involved, and will consider that different variants, antagonists, and binding partners of STD polypeptides may have similar or different effects. For example, an MPD polypeptide or fragment thereof may act as an antagonist of a protein processing function of metalloproteinases (e.g., from other members of the ADAM family of polypeptides) by interacting with an ADAM binding partner and preventing the activity of the metalloproteinase upon its substrate. Accordingly, an MPD may modulate protein processing, such as release of growth factors, adhesion proteins, and inflammatory factors.

[0154] The disclosed ND polypeptides, fragments thereof, antibodies, compositions and combination therapies described herein are useful in medicines for treating bacterial, viral or protozoal infections, and complications resulting therefrom. Cardiovascular disorders are treatable with the disclosed MPD polypeptides, fragments thereof, antibodies, pharmaceutical compositions or combination therapies, including aortic aneurysms; arteritis; vascular occlusion; complications of coronary by-pass surgery; ischemia/reperfusion injury; heart disease; heart failure; and myocardial infarction. In addition, the MPD polypeptides, fragments thereof, antibodies, compositions and combination therapies of the invention can be used to treat chronic pain conditions, to treat various disorders of the endocrine system, conditions of the gastrointestinal system, disorders of the genitourinary system, and anemias and hematological disorders.

[0155] Due to the role of integrins (e.g., α_(v)β₃, α_(v)β₅, β₁, β₄, α₁, α₂, α₃, α₄, α₅, and α₆ integrins) in cell proliferative disorders, including cancer and cancer cell metastasis, also provided herein are methods for using MPD polypeptides, fragments thereof, antibodies, compositions or combination therapies to treat various hematologic and oncologic disorders. For example, soluble MPD disintegrin domains can be used to treat various forms of cancer, including acute myelogenous leukemia, Epstein-Barr virus-positive nasopharyngeal carcinoma, glioma, colon, stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer (SCLC and NSCLC), including cancer-associated cachexia, fatigue, asthenia, paraneoplastic syndrome of cachexia, and hypercalcemia by modulating integrin-associated interactions.

[0156] Additional diseases treatable with the polypeptides, fragments, antibodies, compositions or combination therapies of the invention are solid tumors, including sarcoma, osteosarcoma, and carcinoma, such as adenocarcinoma (e.g., breast cancer) and squamous cell carcinoma. Administration of a soluble MPD disintegrin domain can modulate cell-cell and cell-matrix interactions of such tumor cells and/or modulate the angiogenesis and blood supply to such tumors.

[0157] In addition, the MPD polypeptides, fragments thereof, compositions or combination therapies are useful for treating leukemia, including acute myelogenous leukemia, chronic or acute lymphoblastic leukemia and hairy cell leukemia. Other malignancies with invasive metastatic potential that can be treated with the MPD polypeptides, fragments, antibodies, compositions and combination therapies, include multiple myeloma, various lymphoproliferative disorders such as autoimmune lymphoproliferative syndrome (ALPS), chronic lymphoblastic leukemia, hairy cell leukemia, chronic lymphatic leukemia, peripheral T-cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, Burkitt's lymphoma, Epstein-Barr virus-positive T cell lymphoma, histiocytic lymphoma, Hodgkin's disease, diffuse aggressive lymphoma, acute lymphatic leukemias, T gamma lymphoproliferative disease, cutaneous B cell lymphoma, cutaneous T cell lymphoma (i.e., mycosis fungoides), and Sézary syndrome.

[0158] A combination of at least one MPD polypeptide, fragment thereof, or antibody, and one or more anti-angiogenesis factors or other therapeutic agent(s) may be administered to the subject. The additional therapeutic agent(s) may be administered prior to, concurrently with, or following the administration of the MPD polypeptide, fragment thereof, or antibody. The use of more than one therapeutic agent is particularly advantageous when the subject that is being treated has a solid tumor. In some embodiments of the invention, the treatment further comprises treating the mammal with radiation. Radiation, including brachytherapy and teletherapy, may be administered prior to, concurrently with, or following the administration of the MPD polypeptide, fragment, antibody, or MPD binding partner and/or additional therapeutic agent(s).

[0159] In some embodiments the method includes the administration of, in addition to a MPD polypeptide, fragment thereof, or antibody, one or more therapeutics selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones and antihormones, antibodies, immunotherapeutics, radiotherapeutics, and biological response modifiers.

[0160] In some embodiments the method includes administration of, in addition to an MPD polypeptide, fragment thereof, or antibody, one or more therapeutics selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines and cytokines such as interleukins, interferons (α, β or δ) and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, fluoxymesterone, IL-δ inhibitors, angiostatin, endostatin, kringle 5, angiopoietin-2 or other antagonists of angiopoietin-1, antagonists of platelet-activating factor, antagonists of basic fibroblast growth factor, and COX-2 inhibitors.

[0161] In some embodiments the method includes administration of, in addition to an MPD polypeptide, fragment thereof, or antibody, one or more therapeutic polypeptides, including soluble forms thereof, selected from the group consisting of Fit3 ligand (see, U.S. Pat. No. 5,554,512), CD40 ligand (see, U.S. Pat. No. 5,716,805), IL-2, IL-12, 4-IBB ligand (see, U.S. Pat. No. 5,674,704), anti-4-1BB antibodies, TRAIL, TNF antagonists and TNF receptor antagonists including TNFR/Fc, Tek antagonists, TWEAK antagonists and TWEAK-R (see, U.S. Ser. Nos. 60/172,878 and 60/203,347 and Feng et al., Am. J. Pathol. 156(4):1253) antagonists including TWEAK-R/Fc, VEGF antagonists including anti-VEGF antibodies, VEGF receptor (including VEGF-R1 and VEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists, CD 148 (also referred to as DEP-1, ECRTP, and PTPRJ, see Takahashi et al., J. Am. Soc. Nephrol. 10:213545, 1999; and PCT Publication No. WO 00/15258, 23 Mar. 2000) binding proteins, and nectin-3 (see, Satoh-Horikawa et al., J. Biol. Chem. 275(14):10291, 2000; GenBank accession numbers of human nectin-3 nucleic acid and polypeptide sequences are AF282874 and AAF97597, respectively) antagonists.

[0162] In some preferred embodiments an MPD polypeptide, fragment thereof, or antibody of the invention is used as a component of, or in combination with, “metronomic therapy,” such as that described by Browder et al. and Klement et al. (Cancer Research 60:1878, 2000; J. Clin. Invest. 105(8):R15, 2000; see also Barinaga, Science 288:245, 2000).

[0163] This invention provides compounds, compositions, and methods for treating a subject, preferably a mammalian subject, and most preferably a human subject, who is suffering from a medical disorder and in particular, an MPD-associated disorder. Such MPD-associated disorders include conditions caused (directly or indirectly) or exacerbated by binding between a polypeptide having an MPD sequence and its binding partner (e.g., an integrin). For purposes of this disclosure, the terms “illness,” “disease,” “disorder,” “medical condition,” “abnormal condition” and the like are used interchangeably with the term “medical disorder.” The terms “treat”, “treating”, and “treatment” used herein include curative, preventative (e.g., prophylactic) and palliative or ameliorative treatment. For such therapeutic uses, MPD polypeptides and fragments, MPD polynucleotides encoding an MPD polypeptide, and/or agonists or antagonists of the MPD polypeptide such as antibodies can be administered to the subject in need through known means. Compositions of the invention can contain a polypeptide in any form described herein, such as native polypeptides, variants, derivatives, oligomers, and biologically active fragments. In particular embodiments, the composition comprises a soluble polypeptide or an oligomer comprising soluble MPD polypeptides (e.g., a soluble MPD disintegrin domain).

[0164] In practicing the method of treatment or use of the invention, a therapeutically effective amount of a therapeutic agent of the invention is administered to a subject having a condition to be treated, preferably to treat or ameliorate diseases associated with the activity of an MPD polypeptide. “Therapeutic agent” includes without limitation any MPD polypeptide, fragment, and variant; polynucleotide encoding an MPD polypeptide, fragment, and variant; agonists or antagonists of the an MPD polypeptide such as antibodies; an MPD polypeptide binding partner; complexes formed from an MPD polypeptide, fragment, variant, and binding partner, and the like. As used herein, the term “therapeutically effective amount” means the total amount of each therapeutic agent or other active component of the pharmaceutical composition or method that is sufficient to show a meaningful subject benefit, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual therapeutic agent or active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. As used herein, the phrase “administering a therapeutically effective amount” of a therapeutic agent means that the subject is treated with said therapeutic agent in an amount and for a time sufficient to induce an improvement, and preferably a sustained improvement, in at least one indicator that reflects the severity of the disorder. An improvement is considered “sustained” if the subject exhibits the improvement on at least two occasions separated by one or more weeks. The degree of improvement is determined based on signs or symptoms, and determinations may also employ questionnaires that are administered to the subject, such as quality-of-life questionnaires. Various indicators that reflect the extent of the subject's illness may be assessed for determining whether the amount and time of the treatment is sufficient. The baseline value for the chosen indicator or indicators is established by examination of the subject prior to administration of the first dose of the therapeutic agent. Preferably, the baseline examination is done within about 60 days of administering the first dose. If the therapeutic agent is being administered to treat acute symptoms, the first dose is administered as soon as practically possible after the injury has occurred. Improvement is induced by administering therapeutic agents such as an MPD polypeptide, fragment, antibody, or MPD binding partner until the subject manifests an improvement over baseline for the chosen indicator or indicators. In treating chronic conditions, this degree of improvement is obtained by repeatedly administering this medicament over a period of at least a month or more, e.g., for one, two, or three months or longer, or indefinitely. A period of one to six weeks, or even a single dose, often is sufficient for treating acute conditions. Although the extent of the subject's illness after treatment may appear improved according to one or more indicators, treatment may be continued indefinitely at the same level or at a reduced dose or frequency. Once treatment has been reduced or discontinued, it later may be resumed at the original level if symptoms should reappear.

[0165] One skilled in the art will recognize that suitable dosages will vary, depending upon such factors as the nature and severity of the disorder to be treated, the subject's body weight, age, general condition, and prior illnesses and/or treatments, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices such as standard dosing trials. For example, the therapeutically effective dose can be estimated initially from cell culture assays. The dosage will depend on the specific activity of the compound and can be readily determined by routine experimentation. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (ie., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture, while minimizing tonicities. Such information can be used to more accurately determine useful doses in humans. Ultimately, the attending physician will decide the amount of polypeptide of the invention with which to treat each individual subject. Initially, the attending physician will administer low doses of polypeptide of the invention and observe the subject's response. Larger doses of polypeptide of the invention may be administered until the optimal therapeutic effect is obtained for the subject, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the invention should contain about 0.01 ng to about 100 mg (preferably about 0.1 ng to about 10 mg, more preferably about 0.1 microgram to about 1 mg) of a polypeptide of the invention per kg body weight. In one embodiment of the invention, an MPD polypeptide, fragment, antibody, or MPD binding partner is administered one time per week to treat the various medical disorders disclosed herein. In another embodiment polypeptide, fragment, antibody, or MPD binding partner is administered at least two times per week and in another embodiment at least three times per week. If injected, the effective amount of an MPD polypeptide, fragment, antibody, or MPD binding partner per adult dose ranges from 1-20 mg/m², and preferably is about 5-12 mg/m². Alternatively, a flat dose may be administered whose amount may range from 5-100 mg/dose. Exemplary dose ranges for a flat dose to be administered by subcutaneous injection are 5-25 mg/dose, 25-50 mg/dose and 50-100 mg/dose. In one embodiment of the invention, the various indications described herein are treated by administering a preparation acceptable for injection containing an MPD polypeptide, fragment, antibody, or MPD binding partner at 25 mg/dose, or alternatively, containing 50 mg per dose. The 25 mg or 50 mg dose may be administered repeatedly, particularly for chronic conditions. If a route of administration other than injection is used, the dose is appropriately adjusted in accord with standard medical practices. In many instances, an improvement in a subject's condition will be obtained by injecting a dose of about 25 mg of an MPD polypeptide, fragment, antibody, or MPD binding partner one to three times per week over a period of at least three weeks, or a dose of 50 mg of an MPD polypeptide, fragment, antibody, or MPD binding partner one or two times per week for at least three weeks (a treatment for longer periods may be necessary to induce the desired degree of improvement). For incurable chronic conditions, the regimen may be continued indefinitely, with adjustments being made to dose and frequency if such are deemed necessary by the subject's physician. The foregoing doses are examples for an adult subject who is a person who is 18 years of age or older. For pediatric subjects (age 4-17), a suitable regimen involves the subcutaneous injection of 0.4 mg/kg, up to a maximum dose of 25 mg of an MPD polypeptide, fragment, antibody, or MPD binding partner, administered by subcutaneous injection one or more times per week. If an antibody against an MPD polypeptide is used as an MPD polypeptide antagonist, a preferred dose range is 0.1 to 20 mg/kg, and more preferably is 1-10 mg/kg. Another preferred dose range for an anti-MPD polypeptide antibody is 0.75 to 7.5 mg/kg of body weight. Humanized antibodies are preferred. Such antibodies may be injected or administered intravenously.

[0166] Compositions comprising an effective amount of an MPD polypeptide of the invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources), in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris—HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in Reminigtoni's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, PA. In some embodiments the polypeptide may undergo pegylation to assist in adsorption or uptake. For example, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, so that the characteristics of the carrier will depend on the selected route of administration. In one preferred embodiment of the invention, sustained-release forms of an MPD polypeptide are used. Sustained-release forms suitable for use in the disclosed methods include, but are not limited to, an MPD polypeptide that is encapsulated in a slowly-dissolving biocompatible polymer (such as the alginate microparticles described in U.S. Pat. No. 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant.

[0167] An MPD polypeptide of the invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other polypeptides. As a result, pharmaceutical compositions of the invention may comprise a polypeptide of the invention in such multimeric or complexed form. The pharmaceutical composition of the invention may be in the form of a complex of the polypeptide(s) of invention. The invention further includes the administration of an MPD polypeptide, fragment, antibody, or MPD binding partner concurrently with one or more other drugs that are administered to the same subject in combination, each drug being administered according to a regimen suitable for that medicament. “Concurrent administration” encompasses simultaneous or sequential treatment with the components of the combination, as well as regimens in which the drugs are alternated, or wherein one component is administered long-term and the other(s) are administered intermittently. Components may be administered in the same or in separate compositions, and by the same or different routes of administration. Examples of components that may be included in the pharmaceutical composition of the invention are cytokines, lymphokines, or other hematopoietic factors such as: M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, ILA, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-17, IL-18, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents that either enhance the activity of the polypeptide or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with a polypeptide of the invention, or to minimize side effects. Conversely, an MPD polypeptide, fragment, antibody, or MPD binding partner of the invention may be included in formulations with a particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. Additional examples of drugs to be administered concurrently include but are not limited to antivirals, antibiotics, analgesics, corticosteroids, antagonists of inflammatory cytokines, non-steroidal anti-inflammatories, pentoxifylline, thalidomide, and disease-modifying antiheumatic drugs (DMARDs) such as azathioprine, cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide, minocycline, penicillamine, sulfasalazine and gold compounds such as oral gold, gold sodium thiomalate, and aurothioglucose. Additionally, an MPD polypeptide, fragment, antibody, or MPD binding partner may be combined with a second MPD polypeptide, antibody against an MPD polypeptide, or an MPD polypeptide-derived peptide that acts as a competitive inhibitor of a native an MPD polypeptide.

[0168] Any efficacious route of administration may be used to therapeutically administer an MPD polypeptide, fragment, antibody, or MPD binding partner thereof, including those compositions comprising MPD polynucleotides. Parenteral administration includes injection, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes by bolus injection or by continuous infusion. Other routes include localized administration, e.g., at a site of disease or injury. Other suitable means of administration include sustained release from implants; aerosol inhalation and/or insufflation; eyedrops; vaginal or rectal suppositories; buccal preparations; oral preparations, including pills, syrups, lozenges or chewing gum; and topical preparations such as lotions, gels, sprays, ointments or other suitable techniques. Alternatively, MPD polypeptide, fragment, antibody, or MPD binding partner may be delivered by implanting cells that express the polypeptide, for example, by implanting cells that express an MPD polypeptide, fragment, antibody, or MPD binding partner. Cells may also be cultured ex vivo in the presence of polypeptides of the invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. In another embodiment, the subject's own cells are induced to produce an MPD polypeptide, fragment, antibody, or MPD binding partner by transfection in vivo or ex vivo with a polynucleotide that encodes an MPD polypeptide, fragment, antibody, or MPD binding partner. The polynucleotide can be introduced into the subject's cells, for example, by injecting naked DNA or liposome-encapsulated DNA that encodes an MPD polypeptide, fragment, antibody, or MPD binding partner, or by other means of transfection. Polynucleotides of the invention may also be administered to subjects by other known methods for introduction of nucleic acids into a cell or organism (including, without limitation, in the form of viral vectors).

[0169] When a therapeutically effective amount of an MPD polypeptide, fragment thereof, antibody, or binding partner of the invention is administered orally, the polypeptide will typically be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% a polypeptide of the invention, and preferably from about 25 to 90% a polypeptide of the invention. When adninistered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of a polypeptide of the invention, and preferably from about 1 to 50% a polypeptide of the invention.

[0170] When a therapeutically effective amount of an MPD polypeptide, fragment, antibody, or binding agent of the invention is administered by intravenous, cutaneous or subcutaneous injection, the polypeptide will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable polypeptide solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to a polypeptide of the invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The duration of intravenous therapy using the pharmaceutical composition of the invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual subject. It is contemplated that the duration of each application of a polypeptide of the invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy.

[0171] For compositions of the invention which are useful for tissue repair or regeneration, the therapeutic method includes administering a pyrogen-free, physiologically acceptable form of the composition topically, systematically, locally or in association with an implant or device. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site tissue damage. Additional useful agents may also optionally be included in the composition, as described above, or may be administered simultaneously or sequentially with the composition in the methods of the invention. The compositions can include a matrix capable of delivering the polypeptide-containing composition to the site tissue damage, providing a structure for the developing tissue and optimally capable of being resorbed into the body. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. Potential matrices for the compositions include calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

[0172] In addition to human subjects, compositions comprising an MPD polypeptide, fragment, antibody, or MPD binding partner is useful in the treatment of disease conditions in non-human animals, such as pets (dogs, cats, birds, primates, and the like), domestic farm animals (horses cattle, sheep, pigs, birds, and the like). In such instances, an appropriate dose may be determined according to the animal's body weight. For example, a dose of 0.2-1 mg/kg may be used. Alternatively, the dose is determined according to the animal's surface area, an exemplary dose ranging from 0.1-20 mg/m², or more preferably, from 5-12 mg/m². For small animals, such as dogs or cats, a suitable dose is 0.4 mg/kg. In a one embodiment, an MPD polypeptide, fragment, antibody, or MPD binding partner (preferably constructed from genes derived from the same species as the subject), is administered by injection or other suitable route one or more times per week until the animal's condition is improved, or it may be administered indefinitely.

[0173] The invention also relates to the use an MPD polypeptide, fragment, and variant; polynucleotide encoding an MPD polypeptide, fragment, and variant; agonists or antagonists of an MPD polypeptide such as antibodies; an MPD polypeptide binding partner; complexes formed from an MPD polypeptide, fragment, variant, and binding partner, and the like, in the manufacture of a medicament for the prevention or therapeutic treatment of a disease or disorder.

[0174] Further encompassed by the invention are systems and methods for analyzing NTD polypeptides comprising identifying and/or characterizing one or more MNTD polypeptides, encoding nucleic acids, and corresponding genes, these systems and methods preferably comprising a data set representing a set of one or more MTND molecules, or the use thereof. Accordingly, the invention provides a computer readable medium having stored thereon a member selected from the group consisting of a polypeptide comprising a sequence as set forth in SEQ ID Nos: 1-26, or 27; and a set of polypeptide sequences wherein at least one of said sequences comprises a sequence as set forth in SEQ ID Nos: 1-27.

[0175] One embodiment of the invention comprises a computing environment and a plurality of algorithms selectively executed to analyze a polypeptide or polynucleotide of the invention. Examples of analyses of an ADAM polypeptide include, without limitation, displaying the amino acid sequence of a polypeptide in the set, comparing the amino acid sequence of one polypeptide in the set to the amino acid sequence of another polypeptide in the set, predicting the structure of a polypeptide in the set, determining the nucleotide sequences of nucleic acids encoding a polypeptide in the set, and identifying a gene corresponding to a polypeptide in the set.

[0176] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All headings and subheading provided herein are solely for ease of reading and should not be construed to limit the invention. The terms “a”, “an” and “the” as used herein are meant to encompass the plural unless the context clearly dictates the singular form. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The following examples are intended to illustrate particular embodiments and not to limit the scope of the invention.

EXAMPLE 1

[0177] Identification of MPD Polypeptides

[0178] A data set was received from Celera Genomics (Rockville, Md.) containing amino acid sequences predicted to be encoded by the human genome. This data set was searched using a BLAST algorithm to identify ADAM family polypeptides. The polypeptides set forth in FIG. 1 were identified as having substantially homology to metalloproteinase-disintegrin polypeptides, including the ADAM family of polypeptides.

EXAMPLE 2

[0179] RACE Analysis

[0180] Polynucleotides encoding an MPD polypeptide are identified by rapid amplification of cDNA ends (RACE) analysis. All RACE products are cloned into vectors and sequenced. Sequence analysis of the RACE products provides a number of clones having substantially identical sequences. RACE Analysis kits are available from a number of companies including Roche Molecular Systems. Primers were designed based upon consensus sequences found by RACE product comparison.

[0181] A primer pair comprising nucleotides reverse transcribed from the polypeptide sequences of FIG. 1 are used to PCR amplify a cDNA library from lymph node cells, bone marrow cells, as well as other cell types known in the art. The resulting PCR products are cloned and sequenced using standard protocols.

[0182] An analysis of the MPD sequences provided in FIG. 1 demonstrate that all the sequence contain homology to metalloproteinase and/or disintegrin containing proteins, including members of the ADAM family of proteins. For example, SEQ ID Nos:4-5, 10, 14, 21-22, and 25-26 comprise the sequence HexGHxxGxxHD (SEQ ID NO:28) at residues 208 to 219, 15 to 26, 229 to 240, 555 to 566, 3 to 14, 269 to 280, and 154 to 165, respectively. SEQ ID NO:24 comprises a sequence that has substantial identity to the conserved HexGHxxGxxHD motif. Thus, a polypeptide comprising SEQ ID NO: 4, 5, 10, 14, 21, 22, A4, 25, or 26 is predicted to have metalloproteinase activity. SEQ ID Nos:4, 8, 10, 14, 18-19, 21, and 25, as well as some known metalloproteinases, share homology to a sequence comprising LNIx(I/V)(A/V)LVGLE(V/I)WT and thus a polypeptide comprising SEQ ID NO: 4, 8, 10, 14, 18-19, 21, or 25 is predicted to have metalloproteinase activity. The invention also provides SEQ ID Nos:2, 3, 7, 9, 17, 20, and 27 which have a high degree of homology to the Testicular Metalloproteinase-like, Disintegrin-like, Cysteine rich (TMDC) protein family, including TMDC III, TMDC IVA, and TMDC IVC. Table 1, above, shows the relative identity of representative polypeptides of the invention with TMDC protein family members.

[0183] In addition, to the sequences above having homology to TMDC family members, a number of the polypeptides of FIG. 1 have homology to the ADAM (A Disintegrin And Metalloproteinase) family of proteins. Such polypeptides include metalloproteinase domains characterized as having the highly conserved HExGHxxGxxHD motif (SEQ ID NO:28) and/or a LNIx(I/V)(A/V)LVGLE(V/I)WT motif (SEQ ID NO:29). For example, SEQ ID Nos:4, 10, 14, 21, 25, and 26 comprise a sequence from about amino acids residues 65 to 274 of SEQ ID NO:4; 24 to 235 of SEQ ID NO:10; 85 to 290 of SEQ ID NO:14; 202 to 411 of SEQ ID NO:21; 123 to 332 of SEQ ID NO:25; and 118 to 215 of SEQ ID NO:26.

[0184] A number of polypeptides of FIG. 1 have homology to disintegrin domain sequences typically characterized as containing a conserved motif having a sequence CGN(G/K)×(LN)(E/D)×(G/N)EECDCG (SEQ ID NO:30) (herein after the “CGN-GEEC” motif). For example, SEQ ID Nos:4, 10, 14, 21, and 24-26 contain the CGN-GEEC motif and thus a polypeptide comprising SEQ ID NO: 4, 10, 14, 21, 24, 25, or 26 is predicted to have disintegrin activity. SEQ ID NO: 11 has a putative CGN-GEEC sequence at residues 43 to 57 and thus a polypeptide comprising SEQ ID NO:11 is predicted to have disintegrin activity. In addition, ADAM family of proteins are characterized as having a number of conserved cysteine residues in their disintegrin and cysteine-rich domains. For example, SEQ ID Nos:6, 8, 12, 13, 16, and 23, when aligned with a number of ADAM family members (e.g., ADAM 9 (accession no. NP 003807), align with the conserved cysteine residues in the disintegrin domain of such ADAM family members. Table 2, above, provides a summary of the relative domains and residues characterizing the domains of some of the polypeptides of the invention.

[0185] Variants of the MPD polypeptide sequences can be identified based upon the sequences provided herein. A number of variants are provided herein. Amino acid substitutions and other alterations (deletions, insertions, and the like) to MPD amino acid sequences are predicted to be more likely to alter or disrupt MPD polypeptide activities if they result in changes to the conserved residues of the amino acid sequences as shown in FIG. 1, and particularly if those changes do not substitute an amino acid of similar structure (such as substitution of any one of the aliphatic residues—Ala, Gly, Leu, Ile, or Val—for another aliphatic residue). Conversely, if a change is made to an MPD amino acid sequence resulting in substitution of the residue at that position in an alignment from one of the other MPD polypeptide sequences, it is less likely that such an alteration will affect the function of the altered MPD polypeptide.

EXAMPLE 3

[0186] Monoclonal Antibodies that Bind Polypeptides of the Invention

[0187] A substantially purified MPD polypeptide can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in U.S. Pat. No. 4,411,993. Mice are immunized with an MPD polypeptide immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 μg subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional MPD polypeptide emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for an MPD polypeptide antibody by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of binding of an MPD polypeptide to an MPD polypeptide binding partner.

[0188] Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of an MPD polypeptide in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine myeloma cell line, e.g., NS1 or preferably P3×63Ag8.653 (ATCC CRL 1580). Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymnidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

[0189] The hybridoma cells are screened by ELISA for reactivity against a substantially pure MPD polypeptide by adaptations of the techniques disclosed in Engvall et al., (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. A preferred screening technique is the antibody capture technique described in Beckmann et al., (J. Immunol. 144:4212, 1990). Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti-MPD monoclonal antibody. Alternatively, hybridoma cells can be grown iii vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Polypeptide A or Polypeptide G can also be used, as can chromatography based upon binding to MPD polypeptide.

EXAMPLE 4

[0190] Chromosome Mapping

[0191] The gene corresponding to an MPD polypeptide is mapped using PCR-based mapping strategies. Initial human chromosomal assignments are made using an MPD-specific PCR primers such as those described Example 8 and a BIOS Somatic Cell Hybrid PCRable DNA kit from BIOS Laboratories (New Haven, Conn.), following the manufacturer's instructions. More detailed mapping is performed using a Genebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville, Ala. (see, e.g., Walter, M A et al., Nature Genetics 7:22-28, 1994). Data from this analysis is then submitted electronically to the MIT Radiation Hybrid Mapper (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) following the instructions contained therein. This analysis yields specific genetic marker names which, when submitted electronically to NCBI: (ncbi.nlm.nih.gov/genemap), yield the specific chromosome interval.

EXAMPLE 5

[0192] Activity of MPD-Disintegrin Polypeptides in a Corneal Pocket Assay

[0193] To construct a polynucleotide encoding an MPD disintegrin domain fused to an Fc (an MPDdis-Fc polypeptide), a nucleic acid encoding a disintegrin domain such as amino acid residues of, for example, SEQ ID NO:6; SEQ ID NO:6 from about residue 43 to 148; SEQ ID NO:8 from about residue 1 to 366; SEQ ID NO:8 from about residue 38 to 366; SEQ ID NO:1; SEQ ID NO: 13; SEQ ID NO: 14 from about residue 1 to 622; SEQ ID NO: 14 from about residue 84 to 622; SEQ ID NO: 14 from about residue 299 to 622; SEQ ID NO:16; SEQ ID NO:21 from about residue 1 to 701; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:24 from about residue 278 to 435; SEQ ID NO:25 from about residue 1 to 627; SEQ ID NO:26; or SEQ ID NO:26 from about residue 224 to 383, beginning with the CGN-GEEC sequence and ending prior to a hydrophobic sequence indicative of a transmembrane domain are fused to a nucleic acid sequence encoding an Fc polypeptide. The construct can use the igKappa leader, which is cleaved by the signal peptidase after the C-terminal G (Glycine) amino acid. The soluble form of the molecule is then predicted to start after this residue. A few residues correspoding to codon(s) of the restiction site can be present at either end of the disintegrin domain used to link a disintegrin domain of to the Fc domain.

[0194] A mouse corneal pocket assay is used to quantitate the inhibition of angiogenesis by MPDdis-Fc polypeptides in vivo. In this assay, agents to be tested for angiogenic or antiangiogenic activity are immobilized in a slow release form in a hydron pellet, which is implanted into micropockets created in the corneal epithelium of anesthetized mice. Vascularization is measured as the appearance, density, and extent of vessel in growth from the vascularized corneal limbus into the normally avascular cornea.

[0195] Hydron pellets, as described in Kenyon et al., Invest Opthamol. & Visual Science 37:1625, 1996, incorporate sucralfate with bFGF (90 ng/pellet), bFGF and IgG (11 μg/pellet, control), or bFGF and a range of concentrations of the agent to be tested (e.g., MPDdis-Fc polypeptide). The pellets are surgically implanted into corneal stromal micropockets created by micro-dissection 1 mm medial to the lateral corneal limbus of 6-8 week old male C57BL mice. After five days, at the peak of neovascular response to bFGF, the corneas are photographed using a Zeiss slit lamp at an incipient angle of 35-50° from the polar axis in the meridian containing the pellet. Images are digitized and processed by subtractive color filters (Adobe Photoshop 4.0) to delineate established microvessels by hemoglobin content. Image analysis software (Bioquant, Nashville, Tenn.) is used to calculate the fraction of the corneal image that is vascularized, the vessel density within the vascularized area, and the vessel density within the total cornea. The inhibition of bFGF-induced corneal angiogenesis, as a function of the dose of MPD disintegrin-Fc polypeptide, is determined.

EXAMPLE 6

[0196] Inhibition of Neovascularization by MPD Disintegrin Polypeptides in a Murine Transplant Model

[0197] Survival of heterotopically transplanted cardiac tissue from one mouse donor to the ear skin of another genetically similar mouse requires adequate neovascularization by the transplanted heart and the surrounding tissue, to promote survival and energy for cardiac muscle function. Inadequate vasculature at the site of transplant causes excessive ischemia to the heart, tissue damage, and failure of the tissue to engraft. Agents that antagonize factors involved in endothelial cell migration and vessel formation can decrease angiogenesis at the site of transplant, thereby limiting graft tissue function and ultimately engraftment itself. A murine heterotopic cardiac isograft model is used to demonstrate the antagonistic effects of MPDdis-Fc polypeptides on neovascularization.

[0198] Female BALB/c (≈12 weeks of age) recipients are given neonatal heart grafts from donor mice of the same strain. The donor heart tissue is grafted into the left ear pinnae of the recipient on day 0 and the mice are divided into two groups. The control group receives human IgG (Hu IgG) while the other group receives MPDdis-Fc, both intraperitoneally. The treatments are continued for five consecutive days. The functionality of the grafts is determined by monitoring visible pulsatile activity on days 7 and 14 post-engraftment. The inhibition of functional engraftment, as a function of the dose of MPDdis-Fc, is determined. The histology of the transplanted hearts is examined is order to visualize the effects of MPDdis-Fc on edema at the site of transplant and host and donor tissue vasculature (using, e.g., Factor VIII staining).

EXAMPLE 7

[0199] Treatment of Tumors with MPD Disintegrin (MPDdis) Polypeptides

[0200] MPDdis-Fc are tested in animal models of solid tumors. The effect of the MPDdis-Fc is determined by measuring tumor frequency and tumor growth. The biological activity of MPDdis-Fc is also demonstrated in other in vitro, ex vido, and in vivo assays known in the art, such as calcium mobilization assays and assays to measure platelet activation, recruitment, or aggregation.

EXAMPLE 8

[0201] Tissue Expression of MPD Polypeptides

[0202] Oligonucleotides corresponding to MPD polypeptide coding region can be used to assess MPD mRNA expression using a panel of human tissue and cell line cDNAs (“MTC cDNA,” Clontech). The forward primer and reverse primer are designed to amplify a predicted coding region fragment of a desired length. Tissues and cell lines that expressed MPD mRNA, as evidenced by the presence of an amplified DNA fragment of the desired length are identified. Because an MPD polypeptide of the invention is not expressed in every tissue, the invention provides a method of tissue-typing by utilizing antibodies to the polypeptides of the invention or by utilizing oligonucleotide primers or probes specific for polynucleotides of the invention.

[0203] Accordingly, using a forward primer: 5′-CTGCTGCTGTGGCTGGGAGTG-3′ (SEQ ID NO:43) and a reverse primer: 5′-GTCATACCCAAATTATGACCAAGCTCAGG-3′ (SEQ ID NO:44) the following tissues were found to express an mRNA having a sequence encoding SEQ ID NO:8: placenta, liver, kidney, pancreas, testis stomach, lyph node, heart, lung, colon adenocarcinoma, fetal liver, fetal lung, fetal spleen, fetal skeletal muscle, fetal thymus, fetal kidney, prostate, thymus, ovary, leukocyte, and esophagus.

[0204] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

1 45 1 32 PRT Homo sapiens MISC_FEATURE (1)..(32) X at residue 1 and 32 can be any amino acid 1 Xaa Leu Cys Thr Glu Glu Cys Val His Gly Arg Cys Val Ser Pro Asp 1 5 10 15 Thr Cys His Cys Glu Pro Gly Trp Gly Gly Pro Asp Cys Ser Ser Xaa 20 25 30 2 182 PRT Homo sapiens 2 Met Phe Leu Leu Leu Ala Leu Leu Thr Glu Leu Gly Arg Leu Gln Ala 1 5 10 15 His Glu Gly Ser Glu Gly Ile Phe Leu His Val Thr Val Pro Arg Lys 20 25 30 Ile Lys Ser Asn Asp Ser Glu Val Ser Glu Arg Lys Met Ile Tyr Ile 35 40 45 Ile Thr Ile Asp Gly Gln Pro Tyr Thr Leu His Leu Gly Lys Gln Ser 50 55 60 Phe Leu Pro Gln Asn Phe Leu Val Tyr Thr Tyr Asn Glu Thr Gly Ser 65 70 75 80 Leu His Ser Val Ser Pro Tyr Phe Met Met His Cys His Tyr Gln Gly 85 90 95 Tyr Ala Ala Glu Phe Pro Asn Ser Phe Val Thr Leu Ser Ile Cys Ser 100 105 110 Gly Leu Arg Gly Phe Leu Gln Phe Glu Asn Ile Ser Tyr Gly Ile Glu 115 120 125 Pro Val Glu Ser Ser Ala Arg Phe Glu His Ile Ile Tyr Gln Met Lys 130 135 140 Asn Asn Asp Pro Asn Val Ser Ile Leu Ala Val Asn Tyr Ser His Ile 145 150 155 160 Trp Gln Lys Asp Gln Pro Tyr Lys Val Pro Leu Asn Ser Gln Val Thr 165 170 175 Val Ile Ile Leu Met Leu 180 3 150 PRT Homo sapiens 3 Met Thr Lys Glu Ala Leu Pro Met Asp Gly Pro Tyr Ile Pro Pro Asp 1 5 10 15 Cys Tyr Tyr Leu Gly Tyr Leu Glu Glu Val Pro Gln Ser Met Val Thr 20 25 30 Ile Asp Thr Cys Tyr Gly Gly Leu Arg Gly Ile Met Lys Leu Asp Asp 35 40 45 Leu Ala Tyr Glu Ile Lys Pro Leu Gln Asp Ser Arg Arg Phe Glu His 50 55 60 Val Val Ser Gln Ile Val Ala Glu Pro Asn Ala Thr Val Pro Thr Phe 65 70 75 80 Arg Asp Gly Asp Asn Glu Glu Thr Asp Pro Leu Phe Ser Glu Ala Asn 85 90 95 Asn Ser Met Asn Pro Arg Ile Ser Asn Ser Leu Tyr Ser Ser His Arg 100 105 110 Gly Asn Ile Lys Gly His Val Gln Cys Ser Asn Ser Tyr Tyr His Ile 115 120 125 Tyr Gly Asn Ile Thr Thr Cys Ser Lys Glu Val Val Gln Met Phe Ser 130 135 140 Leu Ile Asp Ser Ile Val 145 150 4 686 PRT Homo sapiens 4 Gly Leu Ile Thr Leu Ser Arg Asn Ala Ser Tyr Tyr Leu Arg Pro Trp 1 5 10 15 Pro Pro Arg Gly Ser Lys Asp Phe Ser Thr His Glu Ile Phe Arg Met 20 25 30 Glu Gln Leu Leu Thr Trp Lys Gly Thr Cys Gly His Arg Asp Pro Gly 35 40 45 Asn Lys Ala Gly Met Thr Ser Leu Pro Gly Gly Pro Gln Ser Arg Gly 50 55 60 Arg Arg Glu Ala Arg Arg Thr Arg Lys Tyr Leu Glu Leu Tyr Ile Val 65 70 75 80 Ala Asp His Thr Leu Phe Leu Thr Arg His Arg Asn Leu Asn His Thr 85 90 95 Lys Gln Arg Leu Leu Glu Val Ala Asn Tyr Val Asp Gln Leu Leu Arg 100 105 110 Thr Leu Asp Ile Gln Val Ala Leu Thr Gly Leu Glu Val Trp Thr Glu 115 120 125 Arg Asp Arg Ser Arg Val Thr Gln Asp Ala Asn Ala Thr Leu Trp Ala 130 135 140 Phe Leu Gln Trp Arg Arg Gly Leu Trp Ala Gln Arg Pro His Asp Ser 145 150 155 160 Ala Gln Leu Leu Thr Gly Arg Ala Phe Gln Gly Ala Thr Val Gly Leu 165 170 175 Ala Pro Val Glu Gly Met Cys Arg Ala Glu Ser Ser Gly Gly Val Ser 180 185 190 Thr Asp His Ser Glu Leu Pro Ile Gly Ala Ala Ala Thr Met Ala His 195 200 205 Glu Ile Gly His Ser Leu Gly Leu Ser His Asp Pro Asp Gly Cys Cys 210 215 220 Val Glu Ala Ala Ala Glu Ser Gly Gly Cys Val Met Ala Ala Ala Thr 225 230 235 240 Gly His Pro Phe Pro Arg Val Phe Ser Ala Cys Ser Arg Arg Gln Leu 245 250 255 Arg Ala Phe Phe Arg Lys Gly Gly Gly Ala Cys Leu Ser Asn Ala Pro 260 265 270 Asp Pro Gly Leu Pro Val Pro Pro Ala Leu Cys Gly Asn Gly Phe Val 275 280 285 Glu Ala Gly Glu Glu Cys Asp Cys Gly Pro Gly Gln Glu Cys Arg Asp 290 295 300 Leu Cys Cys Phe Ala His Asn Cys Ser Leu Arg Pro Gly Ala Gln Cys 305 310 315 320 Ala His Gly Asp Cys Cys Val Arg Cys Leu Leu Lys Pro Ala Gly Ala 325 330 335 Leu Cys Arg Gln Ala Met Gly Asp Cys Asp Leu Pro Glu Phe Cys Thr 340 345 350 Gly Thr Ser Ser His Cys Pro Pro Asp Val Tyr Leu Leu Asp Gly Ser 355 360 365 Pro Cys Ala Arg Gly Ser Gly Tyr Cys Arg Asp Gly Ala Cys Pro Thr 370 375 380 Leu Glu Gln Gln Cys Gln Gln Leu Trp Gly Pro Gly Ser His Pro Ala 385 390 395 400 Pro Glu Ala Cys Phe Gln Val Val Asn Ser Ala Gly Asp Ala His Gly 405 410 415 Asn Cys Gly Gln Asp Ser Glu Gly His Phe Leu Pro Cys Ala Gly Arg 420 425 430 Asp Ala Leu Cys Gly Lys Leu Gln Cys Gln Gly Gly Lys Pro Ser Leu 435 440 445 Leu Ala Pro His Met Val Pro Val Asp Ser Thr Val His Leu Asp Gly 450 455 460 Gln Glu Val Thr Cys Arg Gly Ala Leu Ala Leu Pro Ser Ala Gln Leu 465 470 475 480 Asp Leu Leu Gly Leu Gly Leu Val Glu Pro Gly Thr Gln Cys Gly Pro 485 490 495 Arg Met Val Cys Gln Ser Arg Arg Cys Arg Lys Asn Ala Phe Gln Glu 500 505 510 Leu Gln Arg Cys Leu Thr Ala Cys His Ser His Gly Val Cys Asn Ser 515 520 525 Asn His Asn Cys His Cys Ala Pro Gly Trp Ala Pro Pro Phe Cys Asp 530 535 540 Lys Pro Gly Phe Gly Gly Ser Met Asp Ser Gly Pro Val Gln Ala Glu 545 550 555 560 Asn His Asp Thr Phe Leu Leu Ala Met Leu Leu Ser Val Leu Leu Pro 565 570 575 Leu Leu Pro Gly Ala Gly Leu Ala Trp Cys Cys Tyr Arg Leu Pro Gly 580 585 590 Ala His Leu Gln Arg Cys Ser Trp Gly Cys Arg Arg Asp Pro Ala Cys 595 600 605 Ser Gly Pro Lys Asp Gly Pro His Arg Asp His Pro Leu Gly Gly Val 610 615 620 His Pro Met Glu Leu Gly Pro Thr Ala Thr Gly Gln Pro Trp Pro Leu 625 630 635 640 Ala Pro Gly Ser Pro Ala Asp His Ile His Asn Ile Tyr Pro Pro Pro 645 650 655 Phe Leu Pro Asp Pro Glu Asn Ser His Glu Pro Ser Ser His Pro Glu 660 665 670 Lys Pro Leu Pro Ala Val Ser Pro Asp Pro Gln Gly Gly Ser 675 680 685 5 47 PRT Homo sapiens MISC_FEATURE (47)..(47) X at position 47 can be any amino acid 5 Asp His Ser Glu Asn Ala Ile Gly Val Ala Ala Thr Met Ala His Glu 1 5 10 15 Met Gly His Asn Phe Gly Met Thr His Asp Ser Ala Asp Cys Cys Ser 20 25 30 Ala Ser Ala Ala Asp Gly Gly Cys Ile Met Ala Ala Ala Thr Xaa 35 40 45 6 149 PRT Homo sapiens MISC_FEATURE (149)..(149) X at position 149 can be any amino acid 6 Met Asn Ser Lys Gly Asp Gln Phe Gly Asn Cys Gly Ile Ser Thr Ser 1 5 10 15 Pro Gly Ser Gln Tyr Val Arg Cys Ser Asp Gly Asn Ile Phe Cys Gly 20 25 30 Lys Leu Ile Cys Ser Gly Ile Thr Gly Leu Pro Lys Ile Asn Leu Gln 35 40 45 His Thr Met Ile Gln Val Pro Gln Gly Asp Gly Ser Cys Trp Ser Met 50 55 60 Asp Ala Tyr Met Ser Thr Asp Ile Pro Asp Glu Gly Asp Val His Asn 65 70 75 80 Gly Thr Tyr Cys Ala Pro Asn Lys Val Cys Leu Asn Ser Ala Cys Thr 85 90 95 Asp Lys Thr Pro Val Ile Ser Ala Cys Asn Pro Glu Lys Thr Cys Asn 100 105 110 Gly Lys Gly Val Cys Asn Asp Leu Gly His Cys His Cys Asn Glu Gly 115 120 125 His Ala Pro Pro Asp Cys Val Thr Ala Gly Ser Gly Gly Ser Val Asp 130 135 140 Ser Gly Pro Pro Xaa 145 7 559 PRT Homo sapiens 7 Met Arg Gln Ala Glu Ala Arg Val Thr Leu Arg Ala Pro Leu Leu Leu 1 5 10 15 Leu Gly Leu Trp Val Leu Leu Thr Pro Val Arg Cys Ser Gln Gly His 20 25 30 Pro Ser Trp His Tyr Ala Ser Ser Lys Val Val Ile Pro Arg Lys Glu 35 40 45 Thr His His Gly Lys Asp Leu Gln Phe Leu Gly Trp Leu Ser Tyr Ser 50 55 60 Leu His Phe Gly Gly Gln Arg His Ile Ile His Met Arg Arg Lys His 65 70 75 80 Leu Leu Trp Pro Arg His Leu Leu Val Thr Thr Gln Asp Asp Gln Gly 85 90 95 Ala Leu Gln Met Asp Asp Pro Tyr Ile Pro Pro Asp Cys Tyr Tyr Leu 100 105 110 Ser Tyr Leu Glu Glu Val Pro Leu Ser Met Val Thr Val Asp Met Cys 115 120 125 Cys Gly Gly Leu Arg Gly Ile Met Lys Leu Asp Asp Leu Ala Tyr Glu 130 135 140 Ile Lys Pro Leu Gln Asp Ser Arg Arg Leu Glu His Val Ser Gln Ile 145 150 155 160 Val Ala Glu Pro Asn Ala Thr Gly Pro Thr Phe Arg Asp Gly Asp Asn 165 170 175 Glu Glu Thr Asn Pro Leu Phe Ser Glu Ala Asn Asp Ser Met Asn Pro 180 185 190 Arg Ile Ser Asn Trp Leu Tyr Ser Ser His Arg Gly Asn Ile Lys Gly 195 200 205 Tyr Val Gln Cys Ser Asn Ser Tyr Cys Arg Val Asp Asp Asn Ile Thr 210 215 220 Thr Cys Ser Lys Glu Val Val Gln Met Phe Ser Leu Ser Asp Ser Ile 225 230 235 240 Val Gln Asn Ile Asp Leu Arg Tyr Tyr Ile Tyr Leu Leu Thr Ile Tyr 245 250 255 Asn Asn Cys Asp Pro Ala Pro Val Asn Asp Tyr Arg Val Gln Ser Ala 260 265 270 Met Phe Thr Tyr Phe Arg Thr Thr Phe Phe Asp Thr Phe Arg Val His 275 280 285 Ser Pro Thr Leu Leu Ile Lys Glu Ala Pro His Glu Cys Asn Tyr Glu 290 295 300 Pro Gln Arg Pro Ile Gln Asn Ile Cys Asp Leu Pro Glu Tyr Cys His 305 310 315 320 Gly Thr Thr Val Thr Cys Pro Ala Asn Phe Tyr Met Gln Asp Gly Thr 325 330 335 Pro Cys Thr Glu Glu Gly Tyr Cys Tyr His Gly Asn Cys Thr Asp Arg 340 345 350 Asn Val Leu Cys Lys Val Ile Phe Gly Val Ser Ala Glu Glu Ala Pro 355 360 365 Glu Val Cys Tyr Asp Ile Asn Leu Glu Ser Tyr Arg Phe Gly His Cys 370 375 380 Thr Arg Arg Gln Thr Ala Leu Asn Asn Gln Ala Cys Ala Gly Ile Asp 385 390 395 400 Lys Phe Cys Gly Arg Leu Gln Cys Thr Ser Val Thr His Leu Pro Arg 405 410 415 Leu Gln Glu His Val Ser Phe His His Ser Val Thr Gly Gly Phe Gln 420 425 430 Cys Phe Gly Leu Asp Asp His Arg Ala Thr Asp Thr Thr Asp Val Gly 435 440 445 Cys Val Ile Asp Gly Thr Pro Cys Val His Gly Asn Phe Cys Asn Asn 450 455 460 Thr Arg Cys Asn Ala Thr Ile Thr Ser Leu Gly Tyr Asp Cys Arg Pro 465 470 475 480 Glu Lys Cys Ser His Arg Gly Val Cys Asn Asn Arg Arg Asn Cys His 485 490 495 Cys His Ile Gly Trp Asp Pro Pro Leu Cys Leu Arg Arg Gly Ala Gly 500 505 510 Gly Ser Val Asp Ser Gly Pro Pro Pro Lys Ile Thr Arg Ser Val Lys 515 520 525 Gln Ser Gln Gln Ser Val Met Tyr Leu Arg Val Val Phe Gly Arg Ile 530 535 540 Tyr Thr Phe Ile Ile Ala Leu Leu Phe Gly Met Ala Thr Asn Val 545 550 555 8 366 PRT Homo sapiens 8 Met Arg Ala Val Ser Glu Ala Leu Val His Val Arg Phe Ile His Leu 1 5 10 15 Leu Leu Trp Leu Gly Val Phe Leu Phe Phe Ser Gly Trp Leu Gln Ile 20 25 30 Gly Leu Cys Gln His Gln Ser Leu Pro Glu Val Val Ile Pro Leu Arg 35 40 45 Ile Thr Gly Ala Asp Arg Gly Thr Asp Thr Gln Gly Trp Leu Ser Tyr 50 55 60 Gly Leu Gln Val Gly Ser Gln Ser His Thr Ser Asn Val Gln Glu Glu 65 70 75 80 Val Leu Met Leu Tyr Gln Lys Ala Asp Ala Phe Tyr Ile Pro Leu Gly 85 90 95 Ala Ile Val Thr Val Val Gly Leu Glu Ile Trp Thr Gln Glu Asn Phe 100 105 110 Ile Thr Met Asp Thr Ala Gly Val Gly Leu Lys Glu Ile Cys Lys Trp 115 120 125 Lys Gln Thr Ser Phe Asn Ser Cys Ile Pro His Asp Val Ala His Leu 130 135 140 Ile Val Lys Arg Ser Tyr Gly Ile Thr Leu Gly Leu Ala Asn Val Gly 145 150 155 160 Thr Ile Met Pro Ser Val Glu Val Cys Arg Gln Glu Ser Ser Glu Trp 165 170 175 Asp Leu Pro Glu Trp Phe Asn Gly Thr Ser His Glu Cys Ala Glu Asp 180 185 190 Val Cys Leu Gln Asp Gly Ile Pro Cys Lys Gly Ser Gly His Cys Tyr 195 200 205 Glu Lys Arg Cys Asn Asn Cys Asp Glu Gln Cys Arg Gln Ile Phe Gly 210 215 220 Gln Val Arg Cys Glu Val Thr Asp Ile Leu Cys Gly Arg Val Glu Cys 225 230 235 240 Glu Asn Met Gln Glu Ile Pro Leu Leu Arg Asp His Ser Thr Leu His 245 250 255 Arg Thr His Phe Ile Gly Val Thr Cys Trp Asp Thr Gly Tyr Arg Met 260 265 270 Gly Ile Ser Thr Pro Asp Ile Gly Asp Val Lys Asp Ala Thr Glu Cys 275 280 285 Gly Ser Glu His Val Cys Met His Arg Lys Cys Val Thr Met Ser Leu 290 295 300 Leu Asn Ser Thr Cys Leu Pro Val Thr Cys Asn Met Arg Pro Val Cys 305 310 315 320 Asn Asn Lys His His Cys His Cys Cys Arg Glu Trp Lys Pro Pro Asp 325 330 335 Cys Leu Arg Glu Gly Thr Glu Gly Ser Val Asp Ser Gly Thr Thr Thr 340 345 350 Pro Arg Lys Lys Gly Glu Asn Glu Val Cys Lys Gln Gly Val 355 360 365 9 94 PRT Homo sapiens 9 Met Thr Lys Gly Pro Cys Arg Arg Thr Thr Pro Phe Val Pro Arg Asp 1 5 10 15 Cys Tyr Tyr Asp Cys Tyr Leu Glu Gly Val Pro Gly Ser Val Ala Thr 20 25 30 Leu Asp Thr Cys Arg Gly Gly Leu Arg Gly Met Leu Gln Val Asp Asp 35 40 45 Leu Thr Tyr Glu Ile Lys Pro Leu Glu Ala Phe Ser Lys Phe Glu Tyr 50 55 60 Val Val Ser Leu Leu Val Ser Glu Glu Arg Pro Gly Glu Val Ser Arg 65 70 75 80 Cys Lys Thr Glu Gly Glu Glu Ile Asp Gln Glu Ser Glu Lys 85 90 10 751 PRT Homo sapiens 10 Phe Glu His Ser Lys Pro Thr Thr Arg Asp Trp Ala Leu Gln Phe Thr 1 5 10 15 Gln Gln Thr Lys Lys Arg Pro Arg Arg Met Lys Arg Glu Asp Leu Asn 20 25 30 Ser Met Lys Tyr Val Glu Leu Tyr Leu Val Ala Asp Tyr Leu Glu Phe 35 40 45 Gln Lys Asn Arg Arg Asp Gln Asp Ala Thr Lys His Lys Leu Ile Glu 50 55 60 Ile Ala Asn Tyr Val Asp Lys Phe Tyr Arg Ser Leu Asn Ile Arg Ile 65 70 75 80 Ala Leu Val Gly Leu Glu Val Trp Thr His Gly Asn Met Cys Glu Val 85 90 95 Ser Glu Asn Pro Tyr Ser Thr Leu Trp Ser Phe Leu Ser Trp Arg Arg 100 105 110 Lys Leu Leu Ala Gln Lys Tyr His Asp Asn Ala Gln Leu Ile Thr Gly 115 120 125 Met Ser Phe His Gly Thr Thr Ile Gly Leu Ala Pro Leu Met Ala Met 130 135 140 Cys Ser Val Tyr Gln Ser Gly Gly Val Asn Met Asp His Ser Glu Asn 145 150 155 160 Ala Ile Gly Val Ala Ala Thr Met Ala His Glu Met Gly His Asn Phe 165 170 175 Gly Met Thr His Asp Ser Ala Asp Cys Cys Ser Ala Ser Ala Ala Asp 180 185 190 Gly Gly Cys Ile Met Ala Ala Ala Thr Gly His Pro Phe Pro Lys Val 195 200 205 Phe Asn Gly Cys Asn Arg Arg Glu Leu Asp Arg Tyr Leu Gln Ser Gly 210 215 220 Gly Gly Met Cys Leu Ser Asn Met Pro Asp Thr Arg Met Leu Tyr Gly 225 230 235 240 Gly Arg Arg Cys Gly Asn Gly Tyr Leu Glu Asp Gly Glu Glu Cys Asp 245 250 255 Cys Gly Glu Glu Glu Glu Cys Asn Asn Pro Cys Cys Asn Ala Ser Asn 260 265 270 Cys Thr Leu Arg Pro Gly Ala Glu Cys Ala His Gly Ser Cys Cys His 275 280 285 Gln Cys Lys Leu Leu Ala Pro Gly Thr Leu Cys Arg Glu Gln Ala Arg 290 295 300 Gln Cys Asp Leu Pro Glu Phe Cys Thr Gly Lys Ser Pro His Cys Pro 305 310 315 320 Thr Asn Phe Tyr Gln Met Asp Gly Thr Pro Cys Glu Gly Gly Gln Ala 325 330 335 Tyr Cys Tyr Asn Gly Met Cys Leu Thr Tyr Gln Glu Gln Cys Gln Gln 340 345 350 Leu Trp Gly Pro Gly Ala Arg Pro Ala Pro Asp Leu Cys Phe Glu Lys 355 360 365 Val Asn Val Ala Gly Asp Thr Phe Gly Asn Cys Gly Lys Asp Met Asn 370 375 380 Gly Glu His Arg Lys Cys Asn Met Arg Asp Ala Lys Cys Gly Lys Ile 385 390 395 400 Gln Cys Gln Ser Ser Glu Ala Arg Pro Leu Glu Ser Asn Ala Val Pro 405 410 415 Ile Asp Thr Thr Ile Ile Met Asn Gly Arg Gln Ile Gln Cys Arg Gly 420 425 430 Thr His Val Tyr Arg Gly Pro Glu Glu Glu Gly Asp Met Leu Asp Pro 435 440 445 Gly Leu Val Met Thr Gly Thr Lys Cys Gly Tyr Asn His Ile Cys Phe 450 455 460 Glu Gly Gln Cys Arg Asn Thr Ser Phe Phe Glu Thr Glu Gly Cys Gly 465 470 475 480 Lys Lys Cys Asn Gly His Gly Val Cys Asn Asn Asn Gln Asn Cys His 485 490 495 Cys Leu Pro Gly Trp Ala Pro Pro Phe Cys Asn Thr Pro Gly His Gly 500 505 510 Gly Ser Ile Asp Ser Gly Pro Met Pro Pro Glu Ser Val Gly Pro Val 515 520 525 Val Ala Gly Val Leu Val Ala Ile Leu Val Leu Ala Val Leu Met Leu 530 535 540 Met Tyr Tyr Cys Cys Arg Gln Asn Asn Lys Leu Gly Gln Leu Lys Pro 545 550 555 560 Ser Ala Leu Pro Ser Lys Leu Arg Gln Gln Phe Ser Cys Pro Phe Arg 565 570 575 Val Ser Gln Asn Ser Gly Thr Gly His Ala Asn Pro Thr Phe Lys Leu 580 585 590 Gln Thr Pro Gln Gly Lys Arg Lys Val Phe Leu Asp Leu Cys Val Gln 595 600 605 Val Ile Asn Thr Pro Glu Ile Leu Arg Lys Pro Ser Gln Pro Pro Pro 610 615 620 Arg Pro Pro Pro Asp Tyr Leu Arg Gly Gly Ser Pro Pro Ala Pro Leu 625 630 635 640 Pro Ala His Leu Ser Arg Ala Ala Arg Asn Ser Pro Gly Pro Gly Ser 645 650 655 Gln Ile Glu Arg Thr Glu Ser Ser Arg Arg Pro Pro Pro Ser Arg Pro 660 665 670 Ile Pro Pro Ala Pro Asn Cys Ile Val Ser Gln Asp Phe Ser Arg Pro 675 680 685 Arg Pro Pro Gln Lys Ala Leu Pro Ala Asn Pro Val Pro Gly Arg Arg 690 695 700 Ser Leu Pro Arg Pro Gly Gly Ala Ser Pro Leu Arg Pro Pro Gly Ala 705 710 715 720 Gly Pro Gln Gln Ser Arg Pro Leu Ala Ala Leu Ala Pro Lys Arg Val 725 730 735 Trp Lys Thr Cys Asn Leu Lys Thr Gly Asp Gln Phe Gln Ser Gln 740 745 750 11 127 PRT Homo sapiens MISC_FEATURE (127)..(127) X at position 127 can be any amino acid 11 Gln Ser Asn Gly Val Lys Thr Phe Ser Ser Cys Ser Leu Arg Ser Phe 1 5 10 15 Gln Asn Phe Ile Ser Asn Val Gly Val Lys Cys Leu Gln Asn Lys Pro 20 25 30 Gln Met Gln Lys Lys Ser Pro Lys Pro Val Cys Gly Asn Gly Arg Leu 35 40 45 Glu Gly Asn Glu Ile Cys Asp Cys Gly Thr Glu Ala Ile Leu Gln Ser 50 55 60 Gly Val Glu Cys Arg Pro Lys Ala His Pro Glu Cys Asp Ile Ala Glu 65 70 75 80 Asn Cys Asn Gly Ser Ser Pro Glu Cys Gly Pro Asp Ile Thr Leu Ile 85 90 95 Asn Gly Leu Ser Cys Lys Asn Asn Lys Phe Ile Cys Tyr Asp Gly Asp 100 105 110 Cys His Asp Leu Asp Ala Arg Cys Glu Ser Val Phe Gly Lys Xaa 115 120 125 12 122 PRT Homo sapiens MISC_FEATURE (122)..(122) X at position 122 can be any amino acid 12 Met Asp Leu Ser Leu His Val Ala Ser Leu Cys Leu Pro His His Pro 1 5 10 15 Ser Pro Ala Gln Leu Lys Pro Ala Gly Ala Leu Cys Arg Gln Ala Met 20 25 30 Gly Asp Cys Asp Leu Pro Glu Phe Cys Thr Gly Thr Ser Ser His Cys 35 40 45 Pro Pro Asp Val Tyr Leu Leu Asp Gly Ser Pro Cys Ala Arg Gly Ser 50 55 60 Gly Tyr Cys Trp Asp Gly Ala Cys Pro Thr Leu Glu Gln Gln Cys Gln 65 70 75 80 Gln Leu Trp Gly Pro Gly Ser His Pro Ala Pro Glu Ala Cys Phe Gln 85 90 95 Val Val Asn Ser Ala Gly Asp Ala His Gly Asn Cys Gly Gln Asp Ser 100 105 110 Glu Gly His Phe Leu Pro Cys Ala Arg Xaa 115 120 13 67 PRT Homo sapiens MISC_FEATURE (67)..(67) X at position 67 can be any amino acid 13 Phe Lys Pro Ala Asn Met Ile Cys Arg Lys Ser Val Gly Lys Glu Cys 1 5 10 15 Asp Phe Thr Glu Tyr Cys Asn Gly Asp Leu Pro Tyr Cys Leu Pro Asp 20 25 30 Thr Tyr Val Arg Asp Gly Glu Tyr Cys Asp Ser Gly Gly Ala Phe Cys 35 40 45 Phe Gln Gly Lys Cys Arg Thr Phe Asp Lys Gln Cys Asp Asp Leu Ile 50 55 60 Gly Arg Xaa 65 14 660 PRT Homo sapiens 14 Met Trp Arg Glu Phe Ile Ile His Pro Leu Leu Leu Ala Thr Val Leu 1 5 10 15 Asp Leu Arg Gly Leu Leu His Leu Glu Asn Ala Ser Tyr Gly Ile Glu 20 25 30 Pro Leu Gln Met Ser Ser His Phe Glu His Ile Ile Tyr Arg Met Asp 35 40 45 Asp Val Tyr Lys Glu Pro Leu Lys Cys Gly Val Ser Asn Lys Asp Ile 50 55 60 Glu Lys Glu Thr Ala Lys Asp Glu Glu Glu Glu Pro Pro Ser Met Thr 65 70 75 80 Gln Leu Leu Arg Arg Arg Arg Ala Val Leu Pro Gln Thr Arg Tyr Val 85 90 95 Glu Leu Phe Ile Val Val Asp Lys Glu Arg Tyr Asp Met Met Gly Arg 100 105 110 Asn Gln Thr Ala Val Arg Glu Glu Met Ile Leu Leu Ala Asn Tyr Leu 115 120 125 Asp Ser Met Tyr Ile Met Leu Asn Ile Arg Ile Val Leu Val Gly Leu 130 135 140 Glu Ile Trp Thr Asn Gly Asn Leu Ile Asn Ile Val Gly Gly Ala Gly 145 150 155 160 Asp Val Leu Gly Asn Phe Val Gln Trp Arg Glu Lys Phe Leu Ile Thr 165 170 175 Arg Arg Arg His Asp Ser Ala Gln Leu Val Leu Lys Lys Gly Phe Gly 180 185 190 Gly Thr Ala Gly Met Ala Phe Val Gly Thr Val Cys Ser Arg Ser His 195 200 205 Ala Gly Gly Ile Asn Val Phe Gly Gln Ile Thr Val Glu Thr Phe Ala 210 215 220 Ser Ile Val Ala His Glu Leu Gly His Asn Leu Gly Met Asn His Asp 225 230 235 240 Asp Gly Arg Asp Cys Ser Cys Gly Ala Lys Ser Cys Ile Met Asn Ser 245 250 255 Gly Ala Ser Gly Ser Arg Asn Phe Ser Ser Cys Ser Ala Glu Asp Phe 260 265 270 Glu Lys Leu Thr Leu Asn Lys Gly Gly Asn Cys Leu Leu Asn Ile Pro 275 280 285 Lys Pro Asp Glu Ala Tyr Ser Ala Pro Ser Cys Gly Asn Lys Leu Val 290 295 300 Asp Ala Gly Glu Glu Cys Asp Cys Gly Thr Pro Lys Glu Cys Glu Leu 305 310 315 320 Asp Pro Cys Cys Glu Gly Ser Thr Cys Lys Leu Lys Ser Phe Ala Glu 325 330 335 Cys Ala Tyr Gly Asp Cys Cys Lys Asp Cys Arg Phe Leu Pro Gly Gly 340 345 350 Thr Leu Cys Arg Gly Lys Thr Ser Glu Cys Asp Val Pro Glu Tyr Cys 355 360 365 Asn Gly Ser Ser Gln Phe Cys Gln Pro Asp Val Phe Ile Gln Asn Gly 370 375 380 Tyr Pro Cys Gln Asn Asn Lys Ala Tyr Cys Tyr Asn Gly Met Cys Gln 385 390 395 400 Tyr Tyr Asp Ala Gln Cys Gln Val Ile Phe Gly Ser Lys Ala Lys Ala 405 410 415 Ala Pro Lys Asp Cys Phe Ile Glu Val Asn Ser Lys Gly Asp Arg Phe 420 425 430 Gly Asn Cys Gly Phe Ser Gly Asn Glu Tyr Lys Lys Cys Ala Thr Gly 435 440 445 Leu Ser Leu Lys Phe His Ala Pro Phe Leu Ser Thr Met Leu Gln Glu 450 455 460 Ala Val Arg Gln Thr Gly Thr Tyr Leu Gly Gly Ser Val Cys Cys Met 465 470 475 480 Lys Ser Asp Cys Arg Ile Val Thr Leu Val Lys Asn Ala Leu Cys Gly 485 490 495 Lys Leu Gln Cys Glu Asn Val Gln Glu Ile Pro Val Phe Gly Ile Val 500 505 510 Pro Ala Ile Ile Gln Thr Pro Ser Arg Gly Thr Lys Cys Trp Gly Val 515 520 525 Asp Phe Gln Leu Gly Ser Asp Val Pro Asp Pro Gly Met Val Asn Glu 530 535 540 Gly Thr Lys Cys Gly Ala Gly Lys Ile Cys Arg Asn Phe Gln Cys Val 545 550 555 560 Asp Ala Ser Val Leu Asn Tyr Asp Cys Asp Val Gln Lys Lys Cys His 565 570 575 Gly His Gly Val Cys Asn Ser Asn Lys Asn Cys His Cys Glu Asn Gly 580 585 590 Trp Ala Pro Pro Asn Cys Glu Thr Lys Gly Tyr Gly Gly Ser Val Asp 595 600 605 Ser Gly Pro Thr Tyr Asn Glu Met Asn Thr Ala Leu Arg Asp Gly Leu 610 615 620 Leu Val Phe Phe Phe Leu Ile Val Pro Leu Ile Val Cys Asp Tyr Phe 625 630 635 640 Tyr Leu His Gln Glu Gly Ser Thr Val Glu Lys Leu Leu Gln Lys Glu 645 650 655 Glu Ile Thr Asn 660 15 31 PRT Homo sapiens 15 Glu Cys Glu Leu Ala Pro Cys Cys Glu Gly Ser Thr Cys Lys Leu Lys 1 5 10 15 Ser Phe Ala Glu Cys Ala Tyr Gly Asp Cys Cys Lys Asp Cys Arg 20 25 30 16 60 PRT Homo sapiens 16 Phe Lys Pro Ala Asn Met Ile Cys Arg Lys Ser Val Gly Lys Glu Cys 1 5 10 15 Asp Phe Thr Glu Tyr Cys Asn Gly Asp Leu Pro Tyr Cys Leu Pro Asp 20 25 30 Thr Tyr Val Arg Asp Gly Glu Tyr Cys Asp Ser Gly Gly Ala Phe Cys 35 40 45 Phe Gln Gly Lys Cys Arg Thr Phe Ala Gln Thr Met 50 55 60 17 112 PRT Homo sapiens 17 Val Tyr Cys Phe His Asp Pro Pro Gly Trp Arg Phe Thr Ser Ser Glu 1 5 10 15 Ile Val Ile Pro Arg Lys Val Pro His Lys Arg Gly Gly Val Glu Met 20 25 30 Pro Asp Gln Leu Ser Tyr Ser Met Arg Phe Arg Gly Gln Arg His Val 35 40 45 Ile His Met Lys Leu Lys Lys Asn Met Met Pro Arg His Leu Pro Val 50 55 60 Phe Thr Asp Asn Asp Gln Gly Ala Met Gln Glu Asn Tyr Pro Phe Val 65 70 75 80 Pro Arg Asp Cys Tyr Tyr Asp Cys Tyr Leu Glu Gly Val Pro Gly Ser 85 90 95 Ala Ala Thr Leu Asp Thr Cys Arg Gly Gly Leu His Gly Met Leu Gln 100 105 110 18 57 PRT Homo sapiens MISC_FEATURE (57)..(57) X at position 57 can be any amino acid 18 Phe Tyr Arg Pro Leu Asn Ile Arg Ile Val Leu Val Gly Val Glu Val 1 5 10 15 Trp Asn Asp Met Asp Lys Cys Ser Val Ser Gln Asp Pro Phe Thr Ser 20 25 30 Pro Pro Gln Phe Met Asp Trp Arg Lys Met Lys Leu Leu Pro Arg Lys 35 40 45 Ser His Asp Asn Ala Gln Leu Val Xaa 50 55 19 57 PRT Homo sapiens MISC_FEATURE (57)..(57) X at position 57 can be any amino acid 19 Phe Tyr Arg Pro Leu Asn Ile Arg Ile Val Leu Val Gly Val Glu Val 1 5 10 15 Trp Asn Asp Met Asp Lys Cys Ser Val Ser Gln Asp Pro Phe Thr Ser 20 25 30 Leu His Glu Phe Leu Asp Trp Arg Lys Met Lys Leu Ile Pro Arg Lys 35 40 45 Ser His Asp Asn Ala Gln Leu Val Xaa 50 55 20 168 PRT Homo sapiens 20 Met Thr Asp Val Gly Arg Val Ile Asp Gly Thr Pro Cys Val His Gly 1 5 10 15 Asn Phe Cys Asn Asn Thr Arg Cys Asn Ala Thr Ile Thr Ser Leu Gly 20 25 30 Tyr Asp Cys Arg Leu Glu Lys Cys Ser His Arg Gly Val Cys Asn Asn 35 40 45 Arg Arg Asn Cys His Cys His Ile Gly Trp Asp Pro Pro Leu Cys Leu 50 55 60 Arg Arg Gly Ala Gly Gly Ser Val Asp Ser Gly Pro Pro Pro Lys Arg 65 70 75 80 Thr Cys Ser Leu Arg Gln Ser Gln Gln Ser Glu Met Tyr Leu Arg Val 85 90 95 Val Phe Gly Arg Ile Tyr Ala Phe Ile Ile Ala Leu Leu Phe Gly Thr 100 105 110 Ala Thr Asn Val Gln Thr Tyr Gln Asp His His Arg Ala His Pro Leu 115 120 125 Ser Ala Arg Ala Arg Pro Pro Ser Leu Ala Gln Pro His Ile Val Ser 130 135 140 Gly Pro Val Ser Pro Ser Ser Pro Pro Lys Gln Met Pro Asp Leu Gly 145 150 155 160 His Cys Ser Ala Gln Gly Ala Val 165 21 812 PRT Homo sapiens 21 Met Gly Trp Arg Pro Arg Arg Ala Arg Gly Thr Pro Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Trp Pro Val Pro Gly Ala Gly Val Leu Gln 20 25 30 Gly His Ile Pro Gly Gln Pro Val Thr Pro His Trp Val Leu Asp Gly 35 40 45 Gln Pro Trp Arg Thr Val Ser Leu Glu Glu Pro Val Ser Lys Pro Asp 50 55 60 Met Gly Leu Val Ala Leu Glu Ala Glu Gly Gln Glu Leu Leu Leu Glu 65 70 75 80 Leu Glu Lys Asn His Arg Leu Leu Ala Pro Gly Tyr Ile Glu Thr His 85 90 95 Tyr Gly Pro Asp Gly Gln Pro Val Val Leu Ala Pro Asn His Thr Asp 100 105 110 His Cys His Tyr Gln Gly Arg Val Arg Gly Phe Pro Asp Ser Trp Val 115 120 125 Val Leu Cys Thr Cys Ser Gly Met Ser Gly Leu Ile Thr Leu Ser Arg 130 135 140 Asn Ala Ser Tyr Tyr Leu Arg Pro Trp Pro Pro Arg Gly Ser Lys Asp 145 150 155 160 Phe Ser Thr His Glu Ile Phe Arg Met Glu Gln Leu Leu Thr Trp Lys 165 170 175 Gly Thr Cys Gly His Arg Asp Pro Gly Asn Lys Ala Gly Met Thr Ser 180 185 190 Leu Pro Gly Gly Pro Gln Ser Arg Gly Arg Arg Glu Ala Arg Arg Thr 195 200 205 Arg Lys Tyr Leu Glu Leu Tyr Ile Val Ala Asp His Thr Leu Phe Leu 210 215 220 Thr Arg His Arg Asn Leu Asn His Thr Lys Gln Arg Leu Leu Glu Val 225 230 235 240 Ala Asn Tyr Val Asp Gln Leu Leu Arg Thr Leu Asp Ile Gln Val Ala 245 250 255 Leu Thr Gly Leu Glu Val Trp Thr Glu Arg Asp Arg Ser Arg Val Thr 260 265 270 Gln Asp Ala Asn Ala Thr Leu Trp Ala Phe Leu Gln Trp Arg Arg Gly 275 280 285 Leu Trp Ala Gln Arg Pro His Asp Ser Ala Gln Leu Leu Thr Gly Arg 290 295 300 Ala Phe Gln Gly Ala Thr Val Gly Leu Ala Pro Val Glu Gly Met Cys 305 310 315 320 Arg Ala Glu Ser Ser Gly Gly Val Ser Thr Asp His Ser Glu Leu Pro 325 330 335 Ile Gly Ala Ala Ala Thr Met Ala His Glu Ile Gly His Ser Leu Gly 340 345 350 Leu Ser His Asp Pro Asp Gly Cys Cys Val Glu Ala Ala Ala Glu Ser 355 360 365 Gly Gly Cys Val Met Ala Ala Ala Thr Gly His Pro Phe Pro Arg Val 370 375 380 Phe Ser Ala Cys Ser Arg Arg Gln Leu Arg Ala Phe Phe Arg Lys Gly 385 390 395 400 Gly Gly Ala Cys Leu Ser Asn Ala Pro Asp Pro Gly Leu Pro Val Pro 405 410 415 Pro Ala Leu Cys Gly Asn Gly Phe Val Glu Ala Gly Glu Glu Cys Asp 420 425 430 Cys Gly Pro Gly Gln Glu Cys Arg Asp Leu Cys Cys Phe Ala His Asn 435 440 445 Cys Ser Leu Arg Pro Gly Ala Gln Cys Ala His Gly Asp Cys Cys Val 450 455 460 Arg Cys Leu Leu Lys Pro Ala Gly Ala Leu Cys Arg Gln Ala Met Gly 465 470 475 480 Asp Cys Asp Leu Pro Glu Phe Cys Thr Gly Thr Ser Ser His Cys Pro 485 490 495 Pro Asp Val Tyr Leu Leu Asp Gly Ser Pro Cys Ala Arg Gly Ser Gly 500 505 510 Tyr Cys Trp Asp Gly Ala Cys Pro Thr Leu Glu Gln Gln Cys Gln Gln 515 520 525 Leu Trp Gly Pro Gly Ser His Pro Ala Pro Glu Ala Cys Phe Gln Val 530 535 540 Val Asn Ser Ala Gly Asp Ala His Gly Asn Cys Gly Gln Asp Ser Glu 545 550 555 560 Gly His Phe Leu Pro Cys Ala Gly Arg Asp Ala Leu Cys Gly Lys Leu 565 570 575 Gln Cys Gln Gly Gly Lys Pro Ser Leu Leu Ala Pro His Met Val Pro 580 585 590 Val Asp Ser Thr Val His Leu Asp Gly Gln Glu Val Thr Cys Arg Gly 595 600 605 Ala Leu Ala Leu Pro Ser Ala Gln Leu Asp Leu Leu Gly Leu Gly Leu 610 615 620 Val Glu Pro Gly Thr Gln Cys Gly Pro Arg Met Val Cys Gln Ser Arg 625 630 635 640 Arg Cys Arg Lys Asn Ala Phe Gln Glu Leu Gln Arg Cys Leu Thr Ala 645 650 655 Cys His Ser His Gly Val Cys Asn Ser Asn His Asn Cys His Cys Ala 660 665 670 Pro Gly Trp Ala Pro Pro Phe Cys Asp Lys Pro Gly Phe Gly Gly Ser 675 680 685 Met Asp Ser Gly Pro Val Gln Ala Glu Asn His Asp Thr Phe Leu Leu 690 695 700 Ala Met Leu Leu Ser Val Leu Leu Pro Leu Leu Pro Gly Ala Gly Leu 705 710 715 720 Ala Trp Cys Cys Tyr Arg Leu Pro Gly Ala His Leu Gln Arg Cys Ser 725 730 735 Trp Gly Cys Arg Arg Asp Pro Ala Cys Ser Gly Pro Lys Asp Gly Pro 740 745 750 His Arg Asp His Pro Leu Gly Gly Val His Pro Met Glu Leu Gly Pro 755 760 765 Thr Ala Thr Gly Gln Pro Trp Pro Leu Asp Pro Glu Asn Ser His Glu 770 775 780 Pro Ser Ser His Pro Glu Lys Pro Leu Pro Ala Val Ser Pro Asp Pro 785 790 795 800 Gln Asp Gln Val Gln Met Pro Arg Ser Cys Leu Trp 805 810 22 39 PRT Homo sapiens MISC_FEATURE (39)..(39) X at position 39 can be any amino acid 22 Met Ala His Glu Ile Gly His Ser Leu Gly Leu Ser His Asp Pro Asp 1 5 10 15 Gly Cys Cys Val Glu Ala Ala Ala Glu Ser Gly Gly Cys Val Met Ala 20 25 30 Ala Ala Thr Gly Tyr Ala Xaa 35 23 253 PRT Homo sapiens 23 Met Arg Asp Arg Glu Gln Leu Arg Pro Gly Leu Gly Ala Gly Trp Ala 1 5 10 15 Glu Gly Leu Ala Ser His Gly Glu Pro Glu Asp Gly His Trp Cys Val 20 25 30 Leu Gly Ala Leu Thr His Leu Ser Leu Phe Gly Lys Ser Thr Leu Cys 35 40 45 Thr Glu Glu Cys Val His Gly Arg Cys Val Ser Pro Asp Thr Cys His 50 55 60 Cys Glu Pro Gly Trp Gly Gly Pro Asp Cys Ser Ser Asp Leu Glu Arg 65 70 75 80 Met Gly Arg Trp Glu Leu Leu Leu Ser Pro Met Leu Ser Ser Arg Leu 85 90 95 Ser Arg Leu Lys Gln Leu Gln Met Asn His Arg Lys Ala Ile Thr Leu 100 105 110 Thr Val Pro Arg Val Gln Leu Cys Gln Ala Arg Gly Pro Ser Gln Thr 115 120 125 Arg Leu Gly Arg Leu Leu Pro Ala Ser Val Pro Pro Arg Arg Asn Gly 130 135 140 Lys Gly Glu Glu Ala Gly Ala Gly Glu Pro Arg Pro Cys Gly Ser Pro 145 150 155 160 Asp Leu Cys Gln Asp Glu Asp Gln Ala Gly Lys Gly Gln Pro Met Glu 165 170 175 Thr Pro Glu His Gln Leu Lys Gly Ser Trp Ser Phe Pro Pro His Leu 180 185 190 Tyr Pro Pro Pro Thr His Val Pro Val Met Leu Leu Pro Gly His Ser 195 200 205 Pro Asn Trp Glu Pro Glu Pro Trp Met Val Gln Ile Met Arg Asn Asp 210 215 220 Arg Ser Pro Met Gln Leu Gln Gly Leu Ser Gly Glu Ser Thr Arg His 225 230 235 240 Thr Gly Arg Ser Ala Val Glu Met Ser Asp Ser Leu Ser 245 250 24 435 PRT Homo sapiens 24 Leu Asn Val Ser Thr Ala Val His Arg Gln Gly Ser Lys Thr Ala Ser 1 5 10 15 His Val Pro Ser Ser Gly Ala Ser His Trp Thr Trp Trp Asp Ala Cys 20 25 30 Arg Pro Gln Ser Ile Leu Ser Ser Ala Ser Val Ile Asn Ser Tyr Asp 35 40 45 Glu Asn Asp Ile Arg His Ser Lys Pro Leu Leu Val Gln Lys Thr Asp 50 55 60 Phe Ile Lys Leu Phe Pro Arg Tyr Ile Glu Met His Ile Val Val Asp 65 70 75 80 Lys Asn Leu Tyr Ser Lys Thr Val Thr Leu Glu Gly Phe Ser Val Val 85 90 95 Met Thr Gln Leu Leu Gly Ile Asn Leu Gly Leu Thr Tyr Asp Asp Ile 100 105 110 Tyr Asn Cys Asn Cys Pro Gly Ala Thr Cys Val Met Asn Ser Lys Ala 115 120 125 Met Gly Phe Leu Gln Phe Glu Asn Ile Ser Tyr Gly Ile Glu Pro Val 130 135 140 Glu Ser Ser Ala Arg Phe Glu His Ile Ile Tyr Gln Met Lys Asn Asn 145 150 155 160 Asp Pro Asn Val Ser Ile Leu Ala Val Asn Tyr Ser His Ile Trp Gln 165 170 175 Lys Asp Gln Pro Tyr Lys Val Pro Leu Asn Ser Gln Tyr Pro Asp Ala 180 185 190 Ile Gly Leu Glu Gly Phe Ser Val Ile Ile Ala Gln Leu Leu Gly Leu 195 200 205 Asn Val Gly Leu Thr Tyr Asp Asp Ile Thr Gln Cys Phe Cys Leu Arg 210 215 220 Ala Thr Cys Ile Met Asn His Glu Ala Val Ser Ala Ser Gly Arg Lys 225 230 235 240 Ile Phe Ser Asn Cys Ser Met His Asp Tyr Arg Tyr Phe Val Ser Lys 245 250 255 Phe Glu Thr Lys Cys Leu Gln Lys Leu Ser Asn Leu Gln Pro Leu His 260 265 270 Gln Asn Gln Pro Val Cys Gly Asn Gly Ile Leu Glu Ser Asn Glu Glu 275 280 285 Cys Asp Cys Gly Asn Lys Asn Leu Ser Ile Ala Gly Thr Pro Cys Arg 290 295 300 Lys Ser Ile Asp Pro Glu Cys Asp Phe Thr Glu Tyr Cys Asn Gly Thr 305 310 315 320 Ser Ser Asn Cys Val Pro Asp Thr Tyr Ala Leu Asn Gly Arg Leu Cys 325 330 335 Lys Leu Gly Thr Ala Tyr Cys Tyr Asn Gly Gln Cys Gln Thr Thr Asp 340 345 350 Asn Gln Cys Ala Lys Ile Phe Gly Lys Gly Ala Gln Gly Ala Pro Phe 355 360 365 Ala Cys Phe Lys Glu Val Asn Ser Leu His Glu Arg Ser Glu Asn Cys 370 375 380 Gly Phe Lys Asn Ser Gln Pro Leu Pro Cys Glu Arg Lys Tyr Val Ile 385 390 395 400 Ile Leu Val Ile Val Asn Ala Ser Leu Asp Ile Asp Leu Gln Ile Val 405 410 415 Asn Ser Ser Leu Val Pro Gln Gly Val Val Leu Met Met Glu Ile Phe 420 425 430 Arg Asn Leu 435 25 811 PRT Homo sapiens 25 Gly Ser Glu Glu Gly Ser Pro Lys Leu Gln His Glu Leu Ile Ile Pro 1 5 10 15 Gln Trp Lys Thr Ser Glu Ser Pro Val Arg Glu Lys His Pro Leu Lys 20 25 30 Ala Glu Leu Arg Val Met Ala Glu Gly Arg Glu Leu Ile Leu Asp Leu 35 40 45 Glu Lys Asn Glu Ile Thr Ala Phe Thr Thr Ala Arg Gly Leu Ile Thr 50 55 60 Val Ser Ser Asn Leu Ser Tyr Val Ile Glu Pro Leu Pro Asp Ser Lys 65 70 75 80 Gly Gln His Leu Ile Tyr Arg Ser Glu His Leu Lys Pro Pro Pro Gly 85 90 95 Asn Cys Gly Phe Glu His Ser Lys Pro Thr Thr Arg Asp Trp Ala Leu 100 105 110 Gln Phe Thr Gln Gln Thr Lys Lys Arg Pro Arg Arg Met Lys Arg Glu 115 120 125 Asp Leu Asn Ser Met Lys Tyr Val Glu Leu Tyr Leu Val Ala Asp Tyr 130 135 140 Leu Glu Phe Gln Lys Asn Arg Arg Asp Gln Asp Ala Thr Lys His Lys 145 150 155 160 Leu Ile Glu Ile Ala Asn Tyr Val Asp Lys Phe Tyr Arg Ser Leu Asn 165 170 175 Ile Arg Ile Ala Leu Val Gly Leu Glu Val Trp Thr His Gly Asn Met 180 185 190 Cys Glu Val Ser Glu Asn Pro Tyr Ser Thr Leu Trp Ser Phe Leu Ser 195 200 205 Trp Arg Arg Lys Leu Leu Ala Gln Lys Tyr His Asp Asn Ala Gln Leu 210 215 220 Ile Thr Gly Met Ser Phe His Gly Thr Thr Ile Gly Leu Ala Pro Leu 225 230 235 240 Met Ala Met Cys Ser Val Tyr Gln Ser Gly Gly Val Asn Met Asp His 245 250 255 Ser Glu Asn Ala Ile Gly Val Ala Ala Thr Met Ala His Glu Met Gly 260 265 270 His Asn Phe Gly Met Thr His Asp Ser Ala Asp Cys Cys Ser Ala Ser 275 280 285 Ala Ala Asp Gly Gly Cys Ile Met Ala Ala Ala Thr Gly His Pro Phe 290 295 300 Pro Lys Val Phe Asn Gly Cys Asn Arg Arg Glu Leu Asp Arg Tyr Leu 305 310 315 320 Gln Ser Gly Gly Gly Met Cys Leu Ser Asn Met Pro Asp Thr Arg Met 325 330 335 Leu Tyr Gly Gly Arg Arg Cys Gly Asn Gly Tyr Leu Glu Asp Gly Glu 340 345 350 Glu Cys Asp Cys Gly Glu Glu Glu Glu Cys Asn Asn Pro Cys Cys Asn 355 360 365 Ala Ser Asn Cys Thr Leu Arg Pro Gly Ala Glu Cys Ala His Gly Ser 370 375 380 Cys Cys His Gln Cys Lys Leu Leu Ala Pro Gly Thr Leu Cys Arg Glu 385 390 395 400 Gln Ala Arg Gln Cys Asp Leu Pro Glu Phe Cys Thr Gly Lys Ser Pro 405 410 415 His Cys Pro Thr Asn Phe Tyr Gln Met Asp Gly Thr Pro Cys Glu Gly 420 425 430 Gly Gln Ala Tyr Cys Tyr Asn Gly Met Cys Leu Thr Tyr Gln Glu Gln 435 440 445 Cys Gln Gln Leu Trp Gly Pro Gly Ala Arg Pro Ala Pro Asp Leu Cys 450 455 460 Phe Glu Lys Val Asn Val Ala Gly Asp Thr Phe Gly Asn Cys Gly Lys 465 470 475 480 Asp Met Asn Gly Glu His Arg Lys Cys Asn Met Arg Asp Ala Lys Cys 485 490 495 Gly Lys Ile Gln Cys Gln Ser Ser Glu Ala Arg Pro Leu Glu Ser Asn 500 505 510 Ala Val Pro Ile Asp Thr Thr Ile Ile Met Asn Gly Arg Gln Ile Gln 515 520 525 Cys Arg Gly Thr His Val Tyr Arg Gly Pro Glu Glu Glu Gly Asp Met 530 535 540 Leu Asp Pro Gly Leu Val Met Thr Gly Thr Lys Cys Gly Tyr Asn His 545 550 555 560 Ile Cys Phe Glu Gly Gln Cys Arg Asn Thr Ser Phe Phe Glu Thr Glu 565 570 575 Gly Cys Gly Lys Lys Cys Asn Asp His Gly Val Cys Asn Asn Asn Gln 580 585 590 Asn Cys His Cys Leu Pro Gly Trp Ala Pro Pro Phe Cys Asn Thr Pro 595 600 605 Gly His Gly Gly Ser Ile Asp Ser Gly Pro Met Pro Pro Glu Ser Val 610 615 620 Gly Pro Val Val Ala Gly Val Leu Val Ala Ile Leu Val Leu Ala Val 625 630 635 640 Leu Met Leu Met Tyr Tyr Cys Cys Arg Gln Asn Asn Lys Leu Gly Gln 645 650 655 Leu Lys Pro Ser Ala Leu Pro Ser Lys Leu Arg Gln Gln Phe Arg Val 660 665 670 Ser Gln Asn Ser Gly Thr Gly His Ala Asn Pro Thr Phe Lys Leu Gln 675 680 685 Thr Pro Gln Gly Lys Arg Lys Val Ile Asn Thr Pro Glu Ile Leu Arg 690 695 700 Lys Pro Ser Gln Pro Pro Pro Arg Pro Pro Pro Asp Tyr Leu Arg Gly 705 710 715 720 Leu Leu Gln Ala Ser Ala Ala Pro Glu Gly Thr Pro Gly Lys Pro Ser 725 730 735 Ala Arg Pro Gln Glu Pro Pro Gln Ala Arg Arg Cys Ile Pro Thr Ala 740 745 750 Ala Pro Trp Cys Trp Pro Ser Ala Val Pro Ala Ser Gly Ser Thr Cys 755 760 765 Pro Lys Glu Gly Met Glu Asp Leu Gln Phe Glu Asn Trp Gly Pro Val 770 775 780 Pro Lys Ser Val Ile Val Leu Thr Thr Cys Ile Thr Ala Leu Leu Asp 785 790 795 800 Thr Gln Glu Ser His Gly Asn Ala Asn Trp Lys 805 810 26 383 PRT Homo sapiens 26 Asp Leu Leu Pro Glu Asp Phe Val Val Tyr Thr Tyr Asn Lys Glu Gly 1 5 10 15 Thr Leu Ile Thr Asp His Pro Asn Ile Gln Asn His Cys His Tyr Arg 20 25 30 Gly Tyr Val Glu Gly Val His Asn Ser Ser Ile Ala Leu Ser Asp Cys 35 40 45 Phe Gly Leu Arg Gly Leu Leu His Leu Glu Asn Ala Ser Tyr Gly Ile 50 55 60 Glu Pro Leu Gln Asn Ser Ser His Phe Glu His Ile Ile Tyr Arg Met 65 70 75 80 Asp Asp Val Tyr Lys Glu Pro Leu Lys Cys Gly Val Ser Asn Lys Asp 85 90 95 Ile Glu Lys Glu Thr Ala Lys Asp Glu Glu Glu Glu Pro Pro Ser Met 100 105 110 Thr Gln Leu Leu Arg Arg Arg Arg Ala Val Leu Pro Gln Thr Arg Tyr 115 120 125 Val Glu Leu Phe Ile Val Val Asp Lys Glu Arg Phe Gly Gln Ile Thr 130 135 140 Val Glu Thr Phe Ala Ser Ile Val Ala His Glu Leu Gly His Asn Leu 145 150 155 160 Gly Met Asn His Asp Asp Gly Arg Asp Cys Ser Cys Gly Ala Lys Ser 165 170 175 Cys Ile Met Asn Ser Gly Ala Ser Gly Ser Arg Asn Phe Ser Ser Cys 180 185 190 Ser Ala Glu Asp Phe Glu Lys Leu Thr Leu Asn Lys Gly Gly Asn Cys 195 200 205 Leu Leu Asn Ile Pro Lys Pro Asp Glu Ala Tyr Ser Ala Pro Ser Cys 210 215 220 Gly Asn Lys Leu Val Asp Ala Gly Glu Glu Cys Asp Cys Gly Thr Pro 225 230 235 240 Lys Ser Phe Tyr Val His Arg Asn Val Asn Trp Thr Leu Ala Ala Lys 245 250 255 Glu Val Pro Val Ser Leu Asn His Leu Leu Ser Val His Met Val Thr 260 265 270 Val Val Lys Thr Val Gly Phe Cys Gln Pro Asp Val Phe Ile Gln Asn 275 280 285 Gly Tyr Pro Cys Gln Asn Asn Lys Ala Tyr Cys Tyr Asn Gly Met Cys 290 295 300 Gln Tyr Tyr Asp Ala Gln Cys Gln Val Ile Phe Gly Ser Lys Ala Lys 305 310 315 320 Ala Ala Pro Lys Asp Cys Phe Ile Glu Val Asn Ser Lys Gly Asp Arg 325 330 335 Phe Gly Asn Cys Gly Phe Ser Gly Asn Glu Tyr Lys Lys Cys Ala Thr 340 345 350 Gly Pro Pro Pro Pro Gln Pro Lys Val Ser Ser Gln Gly Asn Leu Ile 355 360 365 Pro Ala Arg Pro Ala Pro Ala Pro Pro Leu Tyr Ser Ser Leu Thr 370 375 380 27 574 PRT Homo sapiens 27 Met Arg Gln Ala Glu Val Arg Val Thr Leu Arg Ala Pro Leu Leu Leu 1 5 10 15 Leu Gly Leu Trp Ala Leu Leu Ala Pro Val Arg Cys Ser Gln Gly Arg 20 25 30 Pro Leu Trp His Tyr Ala Ser Ser Glu Val Val Ile Pro Arg Lys Glu 35 40 45 Thr His His Gly Lys Gly Leu Gln Phe Pro Gly Trp Leu Ser His Ser 50 55 60 Leu Arg Phe Gly Gly Gln Arg His Val Ile His Met Arg Arg Lys His 65 70 75 80 Leu Leu Trp Pro Arg His Leu Leu Val Thr Thr Gln Asp Asp Gln Gly 85 90 95 Ala Leu Pro Met Asp Gly Pro Tyr Ile Pro Pro Asp Cys Tyr Tyr Leu 100 105 110 Gly Tyr Leu Glu Glu Val Pro Gln Ser Met Val Thr Ile Asp Thr Cys 115 120 125 Tyr Gly Gly Leu Arg Gly Ile Met Lys Leu Asp Asp Leu Ala Tyr Glu 130 135 140 Ile Lys Pro Leu Gln Asp Ser Arg Arg Phe Glu His Val Val Ser Gln 145 150 155 160 Ile Val Ala Glu Pro Asn Ala Thr Val Pro Thr Phe Arg Asp Gly Asp 165 170 175 Asn Glu Glu Thr Asp Pro Leu Phe Ser Glu Ala Asn Asn Ser Met Asn 180 185 190 Pro Arg Ile Ser Asn Ser Leu Tyr Ser Ser His Arg Gly Asn Ile Lys 195 200 205 Gly His Val Gln Cys Ser Asn Ser Tyr Tyr His Ile Tyr Gly Asn Ile 210 215 220 Thr Thr Cys Ser Lys Glu Val Val Gln Met Phe Ser Leu Ile Asp Ser 225 230 235 240 Ile Val Gln Asn Ile Asp Leu Trp Tyr Tyr Ile Tyr Leu Leu Thr Ile 245 250 255 Tyr Asn Asn Arg Asp Pro Ala Pro Val Asn Gln Tyr Arg Ile Gln Ser 260 265 270 Ala Met Phe Thr Tyr Phe Lys Thr Thr Phe Phe Asp Thr Phe His Val 275 280 285 His Ser Ser Thr Leu Leu Ile Lys Asp Ala Pro His Glu Ser Asn Tyr 290 295 300 Glu Pro Glu Arg Pro Ile Gln Asn Ile Cys Asp Leu Pro Glu Tyr Cys 305 310 315 320 His Gly Thr Thr Val Thr Cys Pro Ala Asn Phe Tyr Met Gln Asp Gly 325 330 335 Thr Leu Cys Met Glu Glu Gly Tyr Cys Tyr His Gly Asn Cys Thr Asp 340 345 350 Arg Asn Val Leu Cys Lys Ala Met Phe Gly Val Ser Ala Glu Asp Ala 355 360 365 Pro Glu Val Cys Tyr Asp Ile Asn Leu Glu Ser Tyr Arg Phe Gly His 370 375 380 Cys Ile Arg Gln Gln Thr Tyr Leu Ser Tyr Gln Ala Cys Thr Gly Ile 385 390 395 400 Asp Lys Phe Cys Gly Gly Leu Gln Cys Thr Asn Val Thr His Leu Pro 405 410 415 Gln Leu Gln Glu His Val Ser Phe His His Ser Val Arg Gly Gly Phe 420 425 430 Gln Cys Phe Arg Leu Asp Glu His His Ala Thr Asp Met Thr Asp Val 435 440 445 Gly Arg Val Ile Asp Gly Thr Pro Cys Val His Gly Asn Phe Cys Asn 450 455 460 Asn Thr Arg Cys Asn Ala Thr Ile Thr Ser Leu Gly Tyr Asp Cys Arg 465 470 475 480 Leu Glu Lys Cys Ser His Arg Gly Val Cys Asn Asn Arg Arg Asn Cys 485 490 495 His Cys His Ile Gly Trp Asp Pro Pro Leu Cys Leu Arg Arg Gly Ala 500 505 510 Gly Gly Ser Val Asp Ser Gly Pro Pro Pro Lys Arg Thr Arg Ser Leu 515 520 525 Arg Gln Ser Gln Gln Ser Glu Met Tyr Leu Arg Val Val Phe Gly Arg 530 535 540 Ile Tyr Ala Phe Ile Ile Ala Leu Leu Phe Gly Thr Ala Thr Asn Val 545 550 555 560 Gln Thr Tyr Gln Asp His His Arg Leu Gly Arg Asp Ser Tyr 565 570 28 12 PRT Artificial Sequence Conserved Motif Sequence 28 His Glu Xaa Gly His Xaa Xaa Gly Xaa Xaa His Asp 1 5 10 29 14 PRT Artificial Sequence Conserved Motif Sequence 29 Leu Asn Ile Xaa Xaa Xaa Leu Val Gly Leu Glu Xaa Trp Thr 1 5 10 30 16 PRT Artificial Sequence Conserved Motif Sequence 30 Cys Gly Asn Xaa Xaa Xaa Xaa Xaa Gly Asn Glu Glu Cys Asp Cys Gly 1 5 10 15 31 5 PRT Artificial Sequence Linker Moiety 31 Gly Gly Gly Gly Ser 1 5 32 6 PRT Artificial Sequence Linker Moiety 32 Gly Gly Gly Gly Ser Xaa 1 5 33 12 PRT Artificial Sequence Linker Moiety 33 Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser 1 5 10 34 14 PRT Artificial Sequence Linker Moiety 34 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly 1 5 10 35 18 PRT Artificial Sequence Linker Moiety 35 Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Ser Gly Ser Thr 1 5 10 15 Lys Gly 36 18 PRT Artificial Sequence Linker Moiety 36 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly 37 14 PRT Artificial Sequence Linker Moiety 37 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Glu Phe 1 5 10 38 5 PRT Artificial Sequence Localization Sequence 38 Lys Lys Lys Arg Lys 1 5 39 26 PRT Artificial Sequence Localization Sequence 39 Met Leu Phe Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu Gly Ser Thr 20 25 40 4 PRT Artificial Sequence Localization Sequence 40 Lys Asp Glu Leu 1 41 4 PRT Artificial Sequence Localization Sequence 41 Cys Ala Ala Xaa 1 42 4 PRT Artificial Sequence Localization Sequence 42 Cys Cys Xaa Xaa 1 43 21 DNA Artificial Sequence Forward Primer Sequence 43 ctgctgctgt ggctgggagt g 21 44 29 DNA Artificial Sequence Reverse Primer Sequence 44 gtcataccca aattatgacc aagctcagg 29 45 395 PRT Homo sapiens 45 Met Pro Asp Thr Arg Met Leu Tyr Gly Gly Arg Arg Cys Gly Asn Gly 1 5 10 15 Tyr Leu Glu Asp Gly Glu Glu Cys Asp Cys Gly Glu Glu Glu Glu Cys 20 25 30 Asn Asn Pro Cys Cys Asn Ala Ser Asn Cys Thr Leu Arg Pro Gly Ala 35 40 45 Glu Cys Ala His Gly Ser Cys Cys His Gln Cys Lys Leu Leu Ala Pro 50 55 60 Gly Thr Leu Cys Arg Glu Gln Ala Arg Gln Cys Asp Leu Pro Glu Phe 65 70 75 80 Cys Thr Gly Lys Ser Pro His Cys Pro Thr Asn Phe Tyr Gln Met Asp 85 90 95 Gly Thr Pro Cys Glu Gly Gly Gln Ala Tyr Cys Tyr Asn Gly Met Cys 100 105 110 Leu Thr Tyr Gln Glu Gln Cys Gln Gln Leu Trp Gly Pro Gly Ala Arg 115 120 125 Pro Ala Pro Asp Leu Cys Phe Glu Lys Val Asn Val Ala Gly Asp Thr 130 135 140 Phe Gly Asn Cys Gly Lys Asp Met Asn Gly Glu His Arg Lys Cys Asn 145 150 155 160 Met Arg Asp Ala Lys Cys Gly Lys Ile Gln Cys Gln Ser Ser Glu Ala 165 170 175 Arg Pro Leu Glu Ser Asn Ala Val Pro Ile Asp Thr Thr Ile Ile Met 180 185 190 Asn Gly Arg Gln Ile Gln Cys Arg Gly Thr His Val Tyr Arg Gly Pro 195 200 205 Glu Glu Glu Gly Asp Met Leu Asp Pro Gly Leu Val Met Thr Gly Thr 210 215 220 Lys Cys Gly Tyr Asn His Ile Cys Phe Glu Gly Gln Cys Arg Asn Thr 225 230 235 240 Ser Phe Phe Glu Thr Glu Gly Cys Gly Lys Lys Cys Asn Gly His Gly 245 250 255 Val Cys Asn Asn Asn Gln Asn Cys His Cys Leu Pro Gly Trp Ala Pro 260 265 270 Pro Phe Cys Asn Thr Pro Gly His Gly Gly Ser Ile Asp Ser Gly Pro 275 280 285 Met Pro Pro Glu Ser Val Gly Pro Val Val Ala Gly Val Leu Val Ala 290 295 300 Ile Leu Val Leu Ala Val Leu Met Leu Met Tyr Tyr Cys Cys Arg Gln 305 310 315 320 Asn Asn Lys Leu Gly Gln Leu Lys Pro Ser Ala Leu Pro Ser Lys Leu 325 330 335 Arg Gln Gln Phe Arg Val Ser Gln Asn Ser Gly Thr Gly His Ala Asn 340 345 350 Pro Thr Phe Lys Leu Gln Thr Pro Gln Gly Lys Arg Lys Val Ile Asn 355 360 365 Thr Pro Glu Ile Leu Arg Lys Pro Ser Gln Pro Pro Pro Arg Pro Pro 370 375 380 Pro Asp Tyr Leu Arg Asp Ile Ser Ile Arg Arg 385 390 395 

What is claimed is:
 1. A substantially purified polypeptide comprising a sequence selected from the group consisting of: (a) SEQ ID Nos: 3-4, and 6-27; (b) fragments of SEQ ID NO:6 having disintegrin activity; (c) fragments of SEQ ID NO:8 having disintegrin activity; (d) fragments of SEQ ID NO: 14 having disintegrin activity; (e) fragments of SEQ ID NO:24 having metalloproteinase activity; (f) fragments of SEQ ID NO:24 having disintegrin activity; (g) SEQ ID NO:6 from about residue 43 to 148; (h) SEQ ID NO:8 from about residue 1 to 366; (i) SEQ ID NO:8 from about residue 38 to 366; (j) SEQ ID NO: 14 from about residue 1 to 622; (k) SEQ ID NO: 14 from about residue 84 to 622; (l) SEQ ID NO: 14 from about residue 299 to 622; (m) SEQ ID NO:21 from about residue 1 to 701; (n) SEQ ID NO:24 from about residue 1 to 277; (o) SEQ ID NO:24 from about residue 278 to 435; (p) SEQ ID NO:25 from about residue 1 to 332; (q) SEQ ID NO:25 from about residue 1 to 627; (r) SEQ ID NO:26 from about residue 1 to 215; (s) SEQ ID NO:26 from about residue 118 to 215; (t) SEQ ID NO:26 from about residue 224 to 383; and (u) SEQ ID NO:27 from about residue 29 to
 574. 2. A substantially purified polypeptide according to claim 1 having disintegrin activity.
 3. A substantially purified polypeptide according to claim 1 having metalloproteinase activity.
 4. A substantially purified polypeptide comprising a sequence that is at least 97% homologous to a sequence as set forth in SEQ ID NO:3-4, 6-11, 13-18, 20, or 23-27 wherein the polypeptide has a metalloproteinase or disintegrin activity.
 5. A polypeptide of claim 1 linked to a second polypeptide, wherein the second polypeptide is a leucine zipper polypeptide, an Fc polypeptide, or a peptide linker moiety.
 6. An isolated polynucleotide encoding a polypeptide of claim
 1. 7. An expression vector comprising a polynucleotide of claim
 6. 8. A recombinant host cell comprising the polynucleotide of claim
 6. 9. A method for producing a polypeptide, comprising culturing the host cell of claim 8 under conditions promoting expression of the polypeptide.
 10. A polypeptide produced by culturing the host cell of claim 8 under conditions to promote expression of the polypeptide.
 11. A substantially purified antibody that specifically binds to a polypeptide of claim
 1. 12. The antibody of claim 11, wherein the antibody is a monoclonal antibody.
 13. The antibody of claim 11, wherein the antibody is a human or humanized antibody.
 14. A method of designing an inhibitor or binding agent of a polypeptide of claim 1, comprising the steps of determining the three-dimensional structure of the polypeptide, analyzing the three-dimensional structure for binding sites of substrates or ligands, designing a molecule that is predicted to interact with the polypeptide, and determining the inhibitory or binding activity of the molecule.
 15. A method for identifying an agent that modulates an activity of a polypeptide, comprising: (a) contacting the agent with a polypeptide of claim 1 under conditions such that the agent and the polypeptide interact; and (b) determining activity of the polypeptide in the presence of the agent compared to a control, wherein a change in activity is indicative of an agent that modulates the polypeptide's activity.
 16. The method of claim 15, wherein the activity is selected from the group consisting of disintegrin activity, cell adhesion activity, angiogenic activity, metalloproteinase activity, and a combination thereof.
 17. The method of claim 15, wherein the polypeptide has disintegrin activity.
 18. The method of claim 15, wherein the agent is selected from the group consisting of an antibody, a small molecule, a peptide, and a peptidomimetic.
 19. A method for modulating angiogenesis in a cell or mammal, comprising contacting or administering to the cell or mammal a polypeptide of claim 1 in an amount effective to modulate disintegrin activity.
 20. The method of claim 19, wherein the cell is contacted in vitro.
 21. The method of claim 19, wherein the cell is contacted in vivo.
 22. A method for modulating endothelial cell migration, comprising contacting the endothelial cell with a polypeptide of claim
 1. 23. A method of inhibiting the binding of an integrin to a ligand comprising contacting or administering to a cell or mammal that expresses the integrin an effective amount of a polypeptide of claim 2 having disintegrin activity.
 24. The method of claim 23, wherein the mammal is afflicted with a condition selected from the group consisting of ocular disorders; malignant and metastatic conditions; inflammatory diseases; osteoporosis and other conditions mediated by accelerated bone resorption; restenosis; inappropriate platelet activation, recruitment, or aggregation; thrombosis; and a condition requiring tissue repair or wound healing.
 25. The method of claim 19 or 23, wherein the polypeptide is in the form of a multimer.
 26. The method of claim 25, wherein the multimer is a dimer or trimer.
 27. The method of claim 25, wherein the multimer comprises an Fc polypeptide, a leucine zipper, or a peptide linker.
 28. A system for analyzing polypeptides or polynucleotides comprising a data set representing a set of one or more polypeptides of claim 1; a computer; and a computer algorithm in an executable format on the computer for analyzing the polypeptides. 